peerj_json_files/PeerJ_Json_110.json

{
    "v1_Abstract": "Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression , and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison). <?xml:namespace prefix = o ns = \"urn:schemas-microsoft-com:office:office\" />",
    "v1_col_introduction": "introduction  : Until recently it was assumed that prenatal exposure to marijuana is frequently combined with other drugs (e.g. tobacco and alcohol), making cannabis effects difficult to isolate and assess (Kozer and Koren, 2001; Moore et al., 2010). However, the studies of Dekker et al and Hayatbakhsh et al suggested that cannabis use prior to, during the first trimester, or throughout gestation is associated with a higher risk for a low birth weight and neonatal length, as well as preterm labor (Dekker et al., 2012; Hayatbakhsh et al., 2012). Moreover, the presence of the endocannabinoid receptors CB1 and CB2 on placental syncytiotrophoblast (Habayeb et al., 2008a), along with marijuana being the most popular drug of abuse among pregnant population (Brown and Graves, 2013), raised the need to understand the effect of cannabinoids on the\nplacenta .\nCannabidiol (CBD) is one of the most abundant cannabinoids in the marijuana plant (Mechoulam and Shvo, 1963; Mechoulam and Hanus, 2002; Schier et al., 2012). It is a promising candidate for clinical utilization, due to low affinity binding to CB1 and CB2 cannabinoid receptors and no\ncognitive and psychoactive activity (Zuardi, 2008; Deiana, 2012 .(\nPreliminary data from in vitro models indicate that cannabinoids may interact with human P-gp (ABCB1) and BCRP (ABCG2). Acute and long-term exposures to cannabinoids were shown to alter P-gp expression and function (Holland et al., 2006; Zhu et al., 2006). However, BCRP\nexposure to cannabinoids demonstrated functional inhibition only (Holland et al., 2007 .(\nP-gp and BCRP both are thought to be protective for the fetus. These ATP-binding cassette (ABC) efflux transporters expressed at the apical membrane of the polarized syncytiotrophoblast layer (Ni and Mao, 2011), and play a significant role in drug transfer across the placental barrier (Lankas et al., 1998; Mao, 2008; Vahakangas and Myllynen, 2009; Myllynen et al., 2010; Eshkoli et al., 2011). P-gp was found to be an anti-apoptotic cellular agent (Smyth et al., 1998; Huls et al., 2009) and BCRP was shown to have a role in placental tissue and syncytial survival by protecting\n16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40\nPeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013)\nR ev ie w in g M an\nus cr ip t\ncells from pro-apoptotic injuries (Evseenko et al., 2007a; Evseenko et al., 2007b; Hardwick et al.,\n2007; Vahakangas and Myllynen, 2009( .\nWe have already assessed the interaction of CBD with placental BCRP, finding that in both, in vitro and ex vivo systems, CBD inhibited its efflux function (Feinshtein et al., In Press). These results together with other recent findings regarding cannabinoids effect on ABC transporter led us to investigate whether CBD affects placental P-gp on functional and placental P-gp and BCRP on expressional levels (Holland et al., 2006; Zhu et al., 2006; Holland et al., 2007; Holland et al., 2008.( In the present work the implications of CBD exposure on P-gp and BCRP expression and P-gp\nfunction is tested in a human trophoblast-like cell lines BeWo and Jar, as placental model .\n41 42 43 44 45 46 47 48 49 50\nPeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013)\nR ev ie w in g M an\nus cr ip t",
    "v2_Abstract": "Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression in BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison).",
    "v2_col_introduction": "introduction  : Until recently it was assumed that prenatal exposure to marijuana is frequently combined with other drugs (e.g. tobacco and alcohol), making cannabis effects difficult to isolate and assess (Kozer and Koren, 2001; Moore et al., 2010). However, the studies of Dekker et al and Hayatbakhsh et al suggested that cannabis use prior to, during the first trimester, or throughout gestation is associated with a higher risk for a low birth weight and neonatal length, as well as preterm labor (Dekker et al., 2012; Hayatbakhsh et al., 2012). These results, the presence of the endocannabinoid receptors CB1 and CB2 on placental syncytiotrophoblast (Habayeb et al., 2008a), along with marijuana being the most popular drug of abuse among pregnant population (Brown and Graves, 2013), raised the need to\nunderstand the effect of cannabinoids on the placenta .\nThe physiological barrier in the human placenta, separating fetal and maternal tissues, is constructed by the syncytiotrophoblast layer (Ni and Mao, 2011). This polarizer syncytium is rich with active transporters, located either to the apical or basolateral membranes, such as the active ATP-binding cassette (ABC) efflux transporters. They play a significant role in drug transfer across the placental barrier, protecting the fetus and affecting pregnancy outcomes (Lankas et al., 1998; Mao, 2008; Vahakangas and Myllynen, 2009; Myllynen et al., 2010.( ABCB1/P-glycoprotein (P-gp) - a 170 KDa protein, and ABCG2/BCRP (Breast Cancer Resistance Protein) - a 70-72 KDa are both efflux transporters, expressed on the apical side of polarized cells, including biological barriers, secreting organs and stem cells (Allikmets et al., 1998; Doyle et al., 1998; Maliepaard et al., 2001; Zhou, 2008). In the human syncytiotrophoblast they can be detected from the first trimester to term, with gestational age-dependent changes in their expression (Mathias et al., 2005; Sun et al., 2006). They transport a broad variety of structurally diverse compounds, some of which are congruent\n1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25\nPeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013)\nR ev ie w in g M an\nus cr ip t\n3 (Frohlich et al., 2004; Mathias et al., 2005; Zhou, 2008). In addition, BCRP transports a wide range of substrates, including fetal hormonal precursors such as estrone-3-sulfate, naturally occurring carcinogens, porphyries and ceramides (Imai et al., 2003; Krishnamurthy and Schuetz, 2005; Evseenko et al., 2007a; Evseenko et al., 2007b; Mao, 2008; Dietrich et al., 2011). P-gp and BCRP both, are thought to be protective for the fetus. P-gp was found to be an anti-apoptotic cellular agent (Smyth et al., 1998; Huls et al., 2009) and BCRP was shown has a role in placental tissue and syncytial survival by protecting cells from pro-apoptotic injuries (Evseenko et al., 2007a; Evseenko et al., 2007b; Hardwick et al., 2007; Vahakangas\nand Myllynen, 2009( .\nPreliminary data from in vitro models indicate that cannabinoids may interact with human P-gp and BCRP. Acute and chronic exposures to cannabinoids were shown to alter P-gp expression and function (Holland et al., 2006; Zhu et al., 2006). However, BCRP exposure to\ncannabinoids demonstrated functional inhibition only (Holland et al., 2007 .(\nCannabidiol (CBD) is one of the most abundant cannabinoids in the marijuana plant (Mechoulam and Shvo, 1963; Mechoulam and Hanus, 2002; Schier et al., 2012). It is a promising candidate for clinical utilization, due to low affinity binding to CB1 and CB2 cannabinoid receptors and no cognitive and psychoactive activity (Zuardi, 2008; Deiana,\n2012 .(\nThe aim of the present work is to study the implications of CBD exposure on P-gp and BCRP\nexpression in a human trophoblast cell line model - BeWo and Jar cells .\n1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20\nPeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013)\nR ev ie w in g M an\nus cr ip t\n4",
    "v1_text": "materials and methods : Materials : BeWo and Jar cells were obtained from Dr. B. Ugele, Ludwig- Maximilians University, Munich, Germany (Feinshtein et al., 2010; Polachek et al., 2010; Feinshtein et al., In Press). MCF7/P-gp cells (a BCRP expressing and P-gp induced cells), were kindly provided by Prof. Esther Priel (Ben Gurion University, Beer Sheva, Israel)(Feinshtein et al., In Press). All materials for cell culture were purchased from Biological Industries (Israel). CBD was a kind gift from Prof. Raphael Mechoulam (The Hebrew University of Jerusalem, Jerusalem, Israel).Calcein-AM, DiOC2(3), rh123 and cyclosporine A (CsA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A list of all antibodies used in the current research is summarized in supplementary table \u0399. results : CBD impact on BCRP and P-gp protein expression The changes in P-gp and BCRP protein levels in the membrane fraction of BeWo, Jar and MCF7/Pgp cells were studied. Following determination of proper cell fractionation (Figure1A), the basal expression of these two transporters in non-treated cells was determined by Western Blot analysis (Figure 1B). Due to unexpected P-gp expression profile in all three cell lines we verified its basal expression by immunocytochemical fluorescent staining (Figure 1C). It can be seen that our BeWo, Jar and MCF7/P-gp cells express detectible levels of P-gp. For P-gp expression, results of Western Blot were confirmed by immunocytochemistry showing that P-gp expression was the highest in MCF7/P-gp and the lowest in Jar cells. For BCRP (ABCG2), results were confirmed by FACS analysis, showing that BCRP expression was much higher in JAR cells compared to BeWo cells (in full accordance with western blot analysis) (Figure 1D.( The changes in membrane BCRP and P-gp levels in BeWo cells following long-term exposure to CBD are displayed in Figure 2. Indeed, BCRP levels were significantly increased in a concentration-dependent manner (Figure 2B) that was not time-dependent (Figure 2A). Following long-term exposure to CBD, P-gp protein levels significantly decreased, in a concentration-dependent matter (Figure 2D), with no time-dependent effect (Figure 2C). Similarly, in Jar cells (Figure 3), BCRP protein concentrations was significantly elevated following long term exposure to CBD, in a concentration but not time-dependent effect (Figures 3A, B). Due to very low P-gp initial (baseline) expression in the Jar cell line (Figure 1B, C), the expected down-regulation in P-gp expression following long-term exposure to CBD is demonstrated in experiments of 72h treatment only. P-gp is vaguely visualized in the control group while it is almost undetectable in the CBD treated group (Figure 3C). 48h exposure yielded the same results (P-gp levels were almost undetectable \u2013 data not shown), making time and concentration dependent comparisons impractical. 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t To examine whether CBD effects are cell specific, the same experimental routine was applied to MCF/P-gp cells. In these cells BCRP and P-gp behavior under CBD influence was profoundly different from that seen in choriocarcinoma cell lines. P-gp levels dramatically increased following long-term treatment with CBD, in a concentration- but not time-dependent matter (Figure 4A). At the same time, BCRP expression was not affected by CBD (neither time-, nor concentration-dependent effect was observed) (Figure 4B). Thus, we conclude that CBD has a cell-type specific influence upon long-term cellular exposure. CBD impact on MCF7/P-gp cells was not investigated further . CBD impact on BCRP and P-gp mRNA To have a glimpse into the mechanism underlying to the changes seen on the protein level in BCRP and P-gp, we further focused to track the changes that occur on mRNA level of these two transporters. In both, BeWo and Jar cells, BCRP mRNA quantification showed elevation following long-term exposure to CBD (Figures 5A, C), and matched the findings on protein level, supporting transcriptional up-regulation. Likewise, P-gp mRNA expression significantly dropped following long-term treatment with CBD, matching the outcomes seen on the protein level (Figures 5B, D), indicating that the changes in P-gp levels following long-term CBD exposure results from transcriptional down-regulation rather than post-transcriptional changes. figure legends : Figure 1: (A) Cell fractionation: to verify proper fractionation of whole cell lysate, membrane fractions and cytosolic fractions of BeWo, Jar, MCF/P-gp cells were subjected to Western blot analysis. Na+/K+ATPase served as membrane marker, and NF-\u03baB (p65 subunit) served as cytosolic marker. (B) P-gp and BCRP basal expression in MCF7/P-gp, Jar and BeWo cells was determined by Western blotting. (C) Immunocytochemistry: fluorescent staining of BeWo, Jar and MCF7/P-gp cells with anti-P-gp antibody and DAPI. NC \u2013 negative control. (D) BCRP (ABCG2) expression in choriocarcinoma cell lines BeWo and Jar as demonstrated by FACS analysis. Figure 2: Long-term exposure of BeWo cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp - CBD concentration-dependent (D) and time-dependent (C). No statistical significance between 48 and 72h time points. Data is displayed in means \u00b1 s.d. of at least three (n=9) independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test, **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated . Figure 3: Long-term exposure of Jar cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp (C). Data is displayed in means \u00b1 s.d. of at least three (n=12) independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test (Student's t test for (C)), *p<0.05, ***p<0.0001 compared to control or comparison between groups as indicated. 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 4: Long-term exposure of MCF/P-gp cells to CBD: changes in membrane P-gp (A) and BCRP (B) expressed levels. Protein levels are given as percent of control levels. Data is displayed in means \u00b1 s.d. of at least three (n=9) independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test. **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated. Figure 5: Long-term exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. (A), (C) changes in BCRP mRNA in BeWo and Jar cells (respectively). (B), (D) changes in P-gp mRNA in BeWo and Jar cells (respectively). Values are given as fold of change compared to control. Data is displayed as means \u00b1 s.d. of at least three (n=6) independent experiments for each time point. One-Way ANOVA, followed by Bonferroni's multiple comparison test. *p<0.05, **p<0.01, ***p<0.0001 compared to control. Figure 6: Non-cell type specific effect of CBD 25\u00b5M on P-gp dependent calcein and rh123 intracellular efflux in (A) BeWo and (B) MCF7/P-gp cells. Data presented from at least 2 independent experiments (n=33, n=11, respectively), as means \u00b1 s.d. Statistical significance determined by Student's t-test. ***p<0.0001. Figure 7: CBD 10\u00b5M inhibition of P-gp dependent efflux of calcein (A) and DiOC2(3) (B). Data presented from at least 2 independent experiments (n=9, n=6, respectively), as means \u00b1 s.d. Statistical significance determined by One-Way ANOVA, followed by Bonferroni's multiple comparison test for (A) and student's t-test for (B). ***p<0.0001, *p<0.05. 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 1 Cell fractionation, P-gp and BCRP basal expression PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 Long-term exposure of BeWo cells to CBD: changes in P-gp and BCRP protein levels. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 3 Long-term exposure of Jar cells to CBD: changes in P-gp and BCRP protein levels. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 4 Long-term exposure of MCF/P-gp cells to CBD: changes in P-gp and BCRP protein levels. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 5 Long-term exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 6 Non-cell type specific inhibition of P-gp by CBD 25\u00b5M. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Figure 7 Inhibition of P-gp by CBD 10\u00b5M in BeWo cells. PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t acknowledgements : We gratefully thank Prof. Sofia Schreiber-Avissar from the Department of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel, for providing writing assistance. 296 297 298 299 300 301 302 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t References Arnold JC, Hone P, Holland ML and Allen JD (2012) CB2 and TRPV1 receptors mediate cannabinoid actions on MDR1 expression in multidrug resistant cells. Pharmacol Rep 64:751-757. Brown HL and Graves CR (2013) Smoking and marijuana use in pregnancy. Clin Obstet Gynecol 56:107-113. Chen C, Hanson E, Watson JW and Lee JS (2003) P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab. Dispos. 31:312-318. 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Clin. Pharmacol. 50:81-87. Mechoulam R and Hanus L (2002) Cannabidiol: an overview of some chemical and pharmacological aspects. Part I: chemical aspects. Chem. Phys. Lipids 121:35-43. Mechoulam R and Shvo Y (1963) Hashish. I. The structure of cannabidiol. Tetrahedron 19:2073-2078. Minderman H, Vanhoefer U, Toth K, Yin MB, Minderman MD, Wrzosek C, Slovak ML and Rustum YM (1996) DiOC2(3) is not a substrate for multidrug resistance protein (MRP)-mediated drug efflux. Cytometry 25:14-20. Moore DG, Turner JD, Parrott AC, Goodwin JE, Fulton SE, Min MO, Fox HC, Braddick FM, Axelsson EL, Lynch S, Ribeiro H, Frostick CJ and Singer LT (2010) During pregnancy, recreational drug-using women stop taking ecstasy (3,4-methylenedioxy-N-methylamphetamine) and reduce alcohol consumption, but continue to smoke tobacco and cannabis: initial findings from the Development and Infancy Study. J Psychopharmacol 24:1403-1410. Mori N, Iwamoto H, Yokooji T and Murakami T (2012) Characterization of intestinal absorption of quinidine, a P-glycoprotein substrate, given as a powder in rats. Pharmazie 67:384-388. Myllynen P, Kummu M and Sieppi E (2010) ABCB1 and ABCG2 expression in the placenta and fetus: an interspecies comparison. Expert Opin. Drug Metab. Toxicol. 6:1385-1398. Myren M, Mose T, Mathiesen L and Knudsen LE (2007) The human placenta--an alternative for studying foetal exposure. Toxicol. In Vitro 21:1332-1340. 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Nabissi M, Morelli MB, Santoni M and Santoni G (2013) Triggering of the TRPV2 channel by cannabidiol sensitizes glioblastoma cells to cytotoxic chemotherapeutic agents. Carcinogenesis 34:48-57. Ni Z and Mao Q (2011) ATP-binding cassette efflux transporters in human placenta. Curr Pharm Biotechnol 12:674-685. Nieri P, Romiti N, Adinolfi B, Chicca A, Massarelli I and Chieli E (2006) Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells. Br. J. Pharmacol. 148:682-687. Polachek H, Holcberg G, Polachek J, Rubin M, Feinshtein V, Sheiner E and Ben-Zvi Z (2010) Carrier-mediated uptake of Levofloxacin by BeWo cells, a human trophoblast cell line. Arch Gynecol Obstet 281:833-838. Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M and Bates SE (2009) ABCG2: a perspective. Adv. Drug Deliv. Rev. 61:3-13. Schier AR, Ribeiro NP, Silva AC, Hallak JE, Crippa JA, Nardi AE and Zuardi AW (2012) Cannabidiol, a Cannabis sativa constituent, as an anxiolytic drug. Rev Bras Psiquiatr 34 Suppl 1:104-110. Schwarz EB, Moretti ME, Nayak S and Koren G (2008) Risk of hypospadias in offspring of women using loratadine during pregnancy: a systematic review and meta-analysis. Drug Saf. 31:775-788. Scuderi C, Filippis DD, Iuvone T, Blasio A, Steardo A and Esposito G (2009) Cannabidiol in medicine: a review of its therapeutic potential in CNS disorders. Phytother. Res. 23:597-602. Smyth MJ, Krasovskis E, Sutton VR and Johnstone RW (1998) The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis. Proc. Natl. Acad. Sci. U S A 95:7024-7029. Solinas M, Massi P, Cantelmo AR, Cattaneo MG, Cammarota R, Bartolini D, Cinquina V, Valenti M, Vicentini LM, Noonan DM, Albini A and Parolaro D (2012) Cannabidiol inhibits angiogenesis by multiple mechanisms. Br J Pharmacol 167:1218-1231. Sullivan MH (2004) Endocrine cell lines from the placenta. Mol. Cell Endocrinol. 228:103-119. Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG and Gibb W (2006) Expression of the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation. Placenta 27:602-609. 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Sun X and Dey SK (2012) Endocannabinoid signaling in female reproduction. ACS Chem Neurosci 3:349-355. Taylor AH, Ang C, Bell SC and Konje JC (2007) The role of the endocannabinoid system in gametogenesis, implantation and early pregnancy. Hum Reprod Update 13:501-513. Vahakangas K and Myllynen P (2009) Drug transporters in the human blood-placental barrier. Br. J. Pharmacol. 158:665-678. Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG and Gibb W (2006) Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor. Can. J. Physiol. Pharmacol. 84:1251-1258. Zhou SF (2008) Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition. Xenobiotica 38:802-832. Zhu HJ, Wang JS, Markowitz JS, Donovan JL, Gibson BB, Gefroh HA and Devane CL (2006) Characterization of P-glycoprotein inhibition by major cannabinoids from marijuana. J. Pharmacol. Exp. Ther. 317:850-857. Zuardi AW (2008) Cannabidiol: from an inactive cannabinoid to a drug with wide spectrum of action. Rev. Bras. Psiquiatr. 30:271-280. 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t methods : Cell culture and drug treatments: MCF7/P-gp cells were cultured in conditions as previously described (Golan et al., 2009), BeWo and Jar cells were cultured as previously described (Golan et al., 2009; Feinshtein et al., 2010). Briefly, 24h after seeding in 35 x 10 mm cell culture dishes (Corning), growth medium (DMEM for MCF7/P-gp, DMEM/F-12 for BeWo and Jar cells, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 \u00b5g/ml streptomycin and 100 units/ml penicillin in a humidified atmosphere of 95% air and 5% CO2 at 37 \u00b0C) was replaced with fresh one containing CBD 10 or 15\u00b5M (initially dissolved in DMSO) or DMSO (0.08% or 0.12%, respectively, vehicle control). In order to achieve long-term exposure, treatment medium was refreshed every day for 24h, 48h or 72h . Subcellular fractionation: Fractionation procedure was carried out as previously described (Golan et al., 2009), for the isolation of the membrane fraction. The membrane fraction was 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t diluted in sample buffer 1:3 (10% v/v glycerol, 20% v/v SDS 20%, 5% v/v \u03b2-mercaptoethanol, 0.05% w/v bromophenol blue, pH 6.8), boiled for 5 min at 950C (for BCRP determination) or incubated for 30 min at 370C (for P-gp determination) and frozen at -800C until assayed. Aliquots were taken for protein determination using the Lowry assay. Fractions purity was verified using specific markers: Na+/K+ ATPase for membranes and NF/kB p65 for cytosol . discussion : Principal findings of the study: There is a dual effect of CBD on the expression of BCRP and P-gp transporters in trophoblast-like and in MCF7/P-gp cell lines. Under long-term CBD exposure BCRP and P-gp present cell-type specific changes in protein and mRNA levels. Occurring already at the transcriptional level, P-gp protein expression is down-regulated, while that of BCRP is up-regulated. Upon short-term exposure, P-gp efflux function is inhibited by CBD. Cannabinoid effects on pregnancy: Cannabis is under extensive use in western society as a recreational drug. The effect of this drug on pregnancy outcome was under constant debate; however, recently cannabis consumption was reported to be associated with adverse pregnancy outcomes, including preterm birth and fetal growth restriction (Dekker et al., 2012; Hayatbakhsh et al., 2012). Although the mechanisms in which phytocannabinoids exert their effects are not well understood, there is accumulating evidence that endocannabinoids (like anandamide) can influence reproduction, including fertilization, implantation, angiogenesis, embryo development and placental growth (Taylor et al., 2007; Habayeb et al., 2008a; Lewis et al., 2012; Solinas et al., 2012; Sun and Dey, 2012). One possible route to convey the effect of endocannabinoids is through the CB1 and CB2 receptors that are express by the human trophoblast and provide a direct target for cannabinoids (Kenney et al., 1999; Habayeb et al., 2008a; Habayeb et al., 2008b). However, not all endocannabinoids exert their effect through the classic cannabinoids receptors. For example CBD, lacks the central effects of cannabis and works through CB1 and CB2 independent model of action (Scuderi et al., 2009). Since our goal was to test the direct cannabinoid effect on trophoblast transporters BCRP and P-gp, and CBD showed the most potent inhibitory effect (among major cannabinoids) on these transporters (Holland et al., 2006; Zhu et al., 2006; Holland et al., 2007) it made CBD the ideal cannabinoid for the present study. 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t BCRP and P-gp significance in pregnancy: BCRP and P-gp play a key role in transport of drugs and endogenous compounds in the human placenta, affecting the outcome of pregnancy (Robey et al., 2009; Myllynen et al., 2010). They transport a broad variety of structurally diverse compounds, some of which are congruent (Frohlich et al., 2004; Mathias et al., 2005; Zhou, 2008). In addition, BCRP transports a wide range of substrates, including fetal hormonal precursors such as estrone-3-sulfate, naturally occurring carcinogens, porphyries and ceramides (Imai et al., 2003; Krishnamurthy and Schuetz, 2005; Evseenko et al., 2007a; Evseenko et al., 2007b; Mao, 2008; Dietrich et al., 2011). P-gp is expressed in the apical membrane of syncytiotrophoblast and probably is the main placental protective transporter during the first trimester. Its expression (both mRNA and protein) decreases with advancing gestation, being the highest during the first trimester (Gil et al., 2005; Mathias et al., 2005; Sun et al., 2006). BCRP plays an important role as a survival factor in BeWo cells as well as in the human placenta. It is thought to have a protective antiapoptotic role in the trophoblast, regulating their survival under low oxygen conditions (Krishnamurthy and Schuetz, 2006; Yeboah et al., 2006; Evseenko et al., 2007a; Evseenko et al., 2007b .( The trophoblast expression of BCRP may change in different pregnancy complications. Indeed, placentas of women with preterm labor and intra-amniotic inflammation had a higher expression of this transporter than those of women with preterm labor without inflammation. In addition, the mRNA expression of BCRP correlated with that of IL-8, which also increased significantly in placentas of women with preterm labor and inflammation, suggesting that the transfer of drugs across the placenta may be altered in cases of preterm labor with inflammation (Mason et al., 2011 .( CBD impact on BCRP and P-gp upon long-term exposure: In the current study we have demonstrated for the first time that the exposure of trophoblast-like cell lines BeWo and Jar to CBD is associated with two distinct patterns of effects on the expression of the BCRP and P-gp 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t transporters. The use of these cell lines, instead of primary trophoblast cultures, results from the fact that primary trophoblast may rapidly differentiate in culture, continuously changing their gene and protein expression (Evseenko et al., 2006). Comparison of these parameters in regard to CBD influences in such dynamic in vitro environment would be almost impossible. Moreover, BeWo and Jar cell lines, derived from human gestational choriocarcinoma, commonly used as an in vitro model for trophoblast toxicology studies, and they offer a suitable model to study certain aspects of human trophoblast physiology, without the aspect of inter-patient variability (Sullivan, 2004; Khare et al., 2006; Myren et al., 2007.( The first effect that was observed following long-term exposure is that CBD transcriptionally inhibited P-gp membrane expression (as mRNA levels of P-gp were also dramatically reduced following long-term exposure to CBD). The long-term inhibitory effect of CBD on P-gp protein was previously reported in drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) (Holland et al., 2006). The cell specificity of CBD was previously reported (Khare et al., 2006; Hu et al., 2011), yet our study is the first to detect this phenomenon in trophoblast-like cell lines. This observation may have clinical implications, in light of the role of P-gp as one of the key transport mechanisms for numerous drugs in the human placenta, especially during the venerable period of the first trimester in which all the fetal organs are formed. Thus, our results might be especially important to women who use cannabis on a regular basis during the first trimester and are treated with other drugs that are P-gp substrates . The expressional up-regulation of BCRP mRNA and protein in trophoblast-like cell lines following long-term exposure to CBD is the second novel effect reported in our study. This observation is similar to that reported in trophoblast of women with preterm labor and inflammation, suggesting that I) long-term exposure to CBD may elicit an inflammatory response in trophoblast-like cell lines (Mason et al., 2011); and II) that this phenomenon could be a direct compensational consequence of P-gp down-regulation, providing a working defense line to the 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t developing embryo. Interestingly, compensation of such nature was already described in murine placentas (Hutson et al., 2010). Nonetheless, the signal transduction of CBD action in human placenta/trophoblast/choriocarcinoma cells has not been clearly reported yet and needs to be further elucidated . CBD impact on P-gp upon acute exposure: In agreement with previously published data, we found that CBD holds inhibiting properties over P-gp efflux function (Holland et al., 2006; Nieri et al., 2006; Zhu et al., 2006). Moreover, our results, show inhibition of P-gp efflux function in cell lines that naturally express this transporter. It can be seen that CBD 10\u00b5M (Figure. 7) yielded inhibition effects lower than CBD 25\u00b5M (Figure 6). Similarly to results recently presented by us (Feinshtein et al., In Press), this observation could imply that CBD inhibits P-gp in a concentration dependent fashion. However, due to different quantification methods used in present study, this should be further elucidated . Of note, many of the drugs that are prescribed and considered safe to use during pregnancy are in fact P-gp or BCRP substrates, like Loratadine and H2 blockers (i.e. Ranitidine and Cimetidine) (Collett et al., 1999; Chen et al., 2003; Li et al., 2008; Schwarz et al., 2008; Dahan and Amidon, 2009; Gill et al., 2009; Matok et al., 2010). Our finding may have clinical implications, suggesting that the use of cannabis during gestation may alter drug transport through the trophoblast and lead to the absence of a functional placental barrier during the first trimester, leaving the developing embryo unprotected at this vulnerable period of pregnancy. Moreover, the trophoblast of pregnant women exposed to marijuana patients may exhibit some resistance to apoptotic and inflammatory processes due to the effect of CBD on BCRP expression. Conclusions: Following cannabis consumption, all the drugs that are P-gp substrates can potentially penetrate the human placental barrier at higher rates when combined with CBD, and therefore their safety under these conditions is to be questioned. Additionally, changes in placental BCRP expression profile might lead to altered transplacental transport of BCRP 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t substrates, such as medications, naturally occurring carcinogens, hormonal precursors and apoptotic molecules, and influence pregnancy outcomes. valeria feinshtein1*, offer erez2, zvi ben-zvi1, noam erez1, tamar eshkoli2, boaz sheizaf2, : eyal sheiner2, mahmud huleihel3, gershon holcberg2. : 1 - Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. 2 - Department of Obstetrics and Gynecology, Soroka University Medical Center, School of medicine, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84101, Israel. 3 - The Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. *Corresponding author: Valeria Feinshtein M.Med.Sc. Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev . P.O. Box 653, Beer-Sheva, Israel, 84105. Tel.: +972 8 6477373; fax: +972 8 6479303; E-mail: shteiman@bgu.ac.il 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t immunocytochemistry and confocal microscopy : MCF7/P-gp, BeWo and Jar cells were seeded on cover slips and grown to 70% sub-confuency. Cells were then fixed in PFA for 15min and stained with mouse anti-human CD 243 (MDR-1) antibody, diluted in PBS, containing 3% BSA, for 1h at room temperature, followed by incubation with Alexa Fluor - 488 goat anti-mouse antibody in PBS containing 3% BSA, for 1/2h at room temperature. Cover-slips were washed 3 times and mounted onto glass slides with DAPI (4',6-diamidino-2-phenylindole) -containing fluorescent mounting medium (DAPIFluoromount-G, SouthernBiotech ). Immunofluorescence was detected by an Olympus FV-1000 Spectral confocal laserscanning microscope with excitation at 488nm and emission at 520nm. Image analysis was performed using ImageJ v. 1.40C software. FACS analysis of BCRP levels Cells were grown to confluency in 6-well culture dishes, trypsinized, counted, and 0.5 *106 cells were placed in light resistant vials. Cells were washed three times with cold PBS (and each time centrifuged at 1200g for 5 min), and incubated for 30 min with BSA 3% in PBS. Following additional PBS wash, 50\u00b5L of BSA 1% in PBS and antibodies were added (1:10 anti-BCRP FITC conjugated antibody or 1:10 CBL602F FTIC conjugated isotype negative control). Cells were incubated in the dark at 4\u00b0c for 1h, washed with ice cold PBS, re-suspended (in 0.5ml PBS) and analyzed by FACS (BD FACS Vantage.( 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t Immunoblotting Membrane fractions were thawed on the day of assay. Protein aliquots (60-100\u00b5g/lane) were taken for protein separation by SDS-PAGE as previously described (Golan et al., 2009). Semi-quantitative analysis was carried out using a computerized image analysis system (EZQuant-Gel 2.11, EZQuant Biology Software Solutions Ltd., Israel). Equal protein loading was ensured by normalization to Na+/K+ ATPase (for P-gp) or actin (for BCRP .( RNA extraction, reverse transcription, Real-Time Polymerase Chain Reaction (qPCR( Isolation and purification of total RNA from BeWo and Jar cells was carried out using EZ-RNA Kit (Biological Industries, Israel) according to manufacturer's instructions. 1\u00b5g of total RNA was used for reverse transcription using High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA), in 20\u00b5l reaction volume. BCRP and P-gp mRNA was measured by qRT-PCR, as indicated in the manufacturer protocol (Applied Biosystems, Foster City, CA), and performed by the Applied Biosystems Real Time PCR system (7500 system), using TaqMan probes and primers for human BCRP, P-gp and actin (Applied Biosystems, Foster City, CA). The cycling conditions for all primers were as follows: hold for 10 min at 95\u00b0C, followed by 40 cycles consisting of two steps, 15 s at 95\u00b0C (denaturing), and 1 min at 60\u00b0C (annealing-extension). The threshold cycle, which correlates inversely with the mRNA levels of target, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level. P-gp and BCRP mRNA levels were normalized to actin mRNA in the same samples. Results were analyzed by the 2(-Delta-DeltaC(T)) method, demonstrating the relative changes in gene expression from real-time quantitative PCR experiments, using 7500 Software v2.0.4 (Applied Biosystems, Foster City, CA.( 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t P-gp substrate uptake experiments Cells were pre-incubated for 30min with CBD 10 or 25\u00b5M (working concentration previously published (Holland et al., 2006; Ligresti et al., 2006; Zhu et al., 2006; Holland et al., 2007; Holland et al., 2008; De Filippis et al., 2011; Arnold et al., 2012; Harvey et al., 2012; Hill et al., 2012; Maor et al., 2012; Solinas et al., 2012; Dudasova et al., 2013; Juknat et al., 2013; Nabissi et al., 2013), and initially dissolved in DMSO) or CsA 20\u00b5M (known P-gp inhibitor used as positive control (Mori et al., 2012)), dissolved in transport buffer (TB) (pH = 7.4) (Feinshtein et al., 2010), while \u201ccontrol cells\u201d were pre-incubated in TB with the correlating concentration of DMSO. Following pre-incubation, P-gp substrates were added (Calcein-AM or DiOC2(3) or rh123) (Minderman et al., 1996; Martin et al., 2003) and cells were further incubated for 30min. At the end of incubation plates were treated as previously detailed (Feinshtein et al., 2010), and samples were stored (at \u221220\u00b0C) for further analysis. Intracellular fluorescence of all P-gp substrates was quantified by Infinite M200 microplate reader (Tecan) and normalized to protein amount (determined by Lowry method) (Lowry et al., 1951), or detected by an Olympus FV-1000 Spectral confocal laserscanning microscope and analyzed using ImageJ v. 1.40C software. Statistical analysis: All statistics and graphs were carried out using GraphPad Prism5 software. Student's t-test or one-way ANOVA followed by appropriate Bonferroni corrections were used. 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t cbd impact on p-gp function : Only BeWo and MCF7/P-gp cell lines were tested for P-gp inhibition by CBD due to the fact that Jar cells expressed very low P-gp levels. In a preliminary study we observed a non-cell type specific P-gp inhibition by 25\u00b5M CBD in both cell lines, as intracellular calcein and rh123 fluorescence was significantly higher (elevation of 101\u00b150% and 70\u00b130%, respectively) in the presence of CBD (Figure 6A, B). We further examined whether lower concentration of CBD 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t 10\u00b5M also inhibits P-gp, using two different P-gp specific substrates, calcein-AM and DiOC2(3). P-gp was inhibited following short-term (1h) exposure to CBD, as significantly more DiOC2(3) and calcein (elevation of 20\u00b18% and 24\u00b19%, respectively) accumulated in the cells (Figure 7A, B.( 192 193 194 195 196 PeerJ reviewing PDF | (v2013:05:527:1:0:NEW 7 Aug 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "materials and methods : Materials : BeWo and Jar cells were obtained from Dr. B. Ugele, Ludwig- Maximilians University, Munich, Germany. MCF7/P-gp cells (a BCRP expressing and P-gp induced cells), were kindly provided by Prof. Esther Priel (Ben Gurion University, Beer Sheva, Israel). All materials for cell culture were purchased from Biological Industries (Israel). CBD was a kind gift from Prof. Raphael Mechoulam (The Hebrew University of Jerusalem, Jerusalem, Israel). A list of all antibodies used in the current research is summarized in supplementary table \u0399. results : CBD impact on BCRP and P-gp protein expression The changes in P-gp and BCRP protein levels in the membrane fraction of BeWo, Jar and MCF7/Pgp cells were studied. Following determination of proper cell fractionation (Figure. 1A), the basal expression of these two transporters in non-treated cells was determined by Western Blot analysis (Figure. 1B). Due to unexpected P-gp expression profile in all three cell lines we verified its basal expression by immunocytochemical fluorescent staining (Figure. 1C). It can be seen that our BeWo, Jar and MCF7/P-gp cells express detectible levels of P-gp. The results of Western Blot were confirmed by immunocytochemistry showing that P-gp expression was the highest in MCF7/P-gp and the lowest in Jar cells. The changes in membrane BCRP and P-gp levels in BeWo cells following chronic exposure to CBD are displayed in Figure 2. Indeed, BCRP levels were significantly increased in a concentration-dependent manner (Figure. 2B) that was not time-dependent (Figure. 2A). Following chronic exposure to CBD, P-gp protein levels significantly decreased, in a concentration-dependent matter (Figure. 2D), with no time-dependent effect (Figure. 2C). Similarly, in Jar cells (Figure. 3), BCRP protein concentrations was significantly elevated following long term exposure to CBD, in a concentration but not time-dependent effect (Figures. 3A, B). Due to very low P-gp initial (baseline) expression in the Jar cell line (Figure. 1B, C), the expected down-regulation in P-gp expression following chronic exposure to CBD is demonstrated in experiments of 72h treatment only. P-gp is vaguely visualized in the control group while it is almost undetectable in the CBD treated group (Figure. 3C). 48h exposure yielded the same results (P-gp levels were almost undetectable \u2013 data not shown), making time and concentration dependent comparisons impractical. To examine whether CBD effects are cell specific, the same experimental routine was applied to MCF/P-gp cells. In these cells BCRP and P-gp behavior under CBD influence was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 8 profoundly different from that seen in placental cells. P-gp levels dramatically increased following chronic treatment with CBD, in a concentration- but not time-dependent matter (Figure. 4A). At the same time, BCRP expression was not affected by CBD (neither time-, nor concentration-dependent effect was observed) (Figure. 4B). Thus, we conclude that CBD has a cell-type specific influence upon chronic cellular exposure. CBD impact on MCF7/P-gp cells was not investigated further . CBD impact on BCRP and P-gp mRNA To have a glimpse into the mechanism underlying to the changes seen on the protein level in BCRP and P-gp in the human placenta, we further focused to track the changes that occur on mRNA level of these two transporters. In both, BeWo and Jar cells, BCRP mRNA quantification showed elevation following chronic exposure to CBD (Figures. 5A, C), and matched the findings on protein level, supporting transcriptional up-regulation. Likewise, P-gp mRNA expression significantly dropped following chronic treatment with CBD, matching the outcomes seen on the protein level (Figures. 5B, D), indicating that the changes in P-gp levels following chronic CBD exposure results from transcriptional down-regulation rather than post-transcriptional changes. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 9 figure legends : Figure 1: (A) Cell fractionation: to verify proper fractionation of whole cell lysate, membrane fractions and cytosolic fractions of BeWo, Jar, MCF/P-gp cells were subjected to Western blot analysis. Na+/K+ATPase served as membrane marker, and NF-\u03baB (p65 subunit) served as cytosolic marker. (B) P-gp and BCRP basal expression in MCF7/P-gp, Jar and BeWo cells was determined by Western blotting. (C) Immunocytochemistry: fluorescent staining of BeWo, Jar and MCF7/P-gp cells with anti-P-gp antibody and DAPI . Figure 2: Chronic exposure of BeWo cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp - CBD concentration-dependent (D) and time-dependent (C). No statistical significance between 48 and 72h time points. Data is displayed in means \u00b1 s.d. of at least three independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test, **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated . Figure 3: Chronic exposure of Jar cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp (C). Data is displayed in means \u00b1 s.d. of at least three independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test (Student's t test for (C)), *p<0.05, ***p<0.0001 compared to control or comparison between groups as indicated. Figure 4: Chronic exposure of MCF/P-gp cells to CBD: changes in membrane P-gp (A) and BCRP (B) expressed levels. Protein levels are given as percent of control levels. Data is displayed in means \u00b1 s.d. of at least three independent experiments of each concentration or 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 20 time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test. **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated. Figure 5: Chronic exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. (A), (C) changes in BCRP mRNA in BeWo and Jar cells (respectively). (B), (D) changes in P-gp mRNA in BeWo and Jar cells (respectively). Values are given as fold of change compared to control. Data is displayed as means \u00b1 s.d. of at least three independent experiments for each time point. One-Way ANOVA, followed by Bonferroni's multiple comparison test. *p<0.05, **p<0.01, ***p<0.0001 compared to control. 1 2 3 4 5 6 7 8 9 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t Figure 1 Cell fractionation; BCRP and P-gp basal expression in BeWo, Jar and MCF7/P-gp cells. (A) Cell fractionation: to verify proper fractionation of whole cell lysate, membrane fractions and cytosolic fractions of BeWo, Jar, MCF/P-gp cells were subjected to Western blot analysis. Na + /K + ATPase served as membrane marker, and NF-\u03baB (p65 subunit) served as cytosolic marker. (B) P-gp and BCRP basal expression in MCF7/P-gp, Jar and BeWo cells was determined by Western blotting. (C) Immunocytochemistry: fluorescent staining of BeWo, Jar and MCF7/P-gp cells with anti-P-gp antibody and DAPI. PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t Figure 2 Chronic exposure of BeWo cells to CBD: changes in membrane BCRP and P-gp expressed levels. Chronic exposure of BeWo cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp - CBD concentration-dependent (D) and time-dependent (C). No statistical significance between 48 and acknowledgements : We gratefully thank Prof. Sofia Schreiber-Avissar from the Department of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel, for providing writing assistance. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 13 References Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V and Dean M (1998) A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res 58:5337-5339. Brown HL and Graves CR (2013) Smoking and marijuana use in pregnancy. Clin Obstet Gynecol 56:107-113. Chen C, Hanson E, Watson JW and Lee JS (2003) P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab. Dispos. 31:312-318. 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Expert Opin. Drug Metab. Toxicol. 6:1385-1398. Myren M, Mose T, Mathiesen L and Knudsen LE (2007) The human placenta--an alternative for studying foetal exposure. Toxicol. In Vitro 21:1332-1340. Ni Z and Mao Q (2011) ATP-binding cassette efflux transporters in human placenta. Curr Pharm Biotechnol 12:674-685. Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M and Bates SE (2009) ABCG2: a perspective. Adv. Drug Deliv. Rev. 61:3-13. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 17 Schier AR, Ribeiro NP, Silva AC, Hallak JE, Crippa JA, Nardi AE and Zuardi AW (2012) Cannabidiol, a Cannabis sativa constituent, as an anxiolytic drug. Rev Bras Psiquiatr 34 Suppl 1:104-110. Schwarz EB, Moretti ME, Nayak S and Koren G (2008) Risk of hypospadias in offspring of women using loratadine during pregnancy: a systematic review and meta-analysis. Drug Saf. 31:775-788. 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Xenobiotica 38:802-832. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 18 Zhu HJ, Wang JS, Markowitz JS, Donovan JL, Gibson BB, Gefroh HA and Devane CL (2006) Characterization of P-glycoprotein inhibition by major cannabinoids from marijuana. J. Pharmacol. Exp. Ther. 317:850-857. Zuardi AW (2008) Cannabidiol: from an inactive cannabinoid to a drug with wide spectrum of action. Rev. Bras. Psiquiatr. 30:271-280. 1 2 3 4 5 6 7 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 19 discussion : Principal findings of the study: There is a dual effect of CBD on the expression of BCRP and P-gp transporters in trophoblast and in MCF7/P-gp cell lines. Under chronic CBD exposure BCRP and P-gp present cell-type specific changes in protein and mRNA levels. Occurring already at the transcriptional level, P-gp protein expression is down-regulated, while that of BCRP is up-regulated. Cannabis is under extensive use in western society as a recreational drug. The effect of this drug on pregnancy outcome was under constant debate; however, recently cannabis consumption was reported to be associated with adverse pregnancy outcomes, including preterm birth and fetal growth restriction (Dekker et al., 2012; Hayatbakhsh et al., 2012). Although the mechanisms in which phytocannabinoids exert their effects are not well understood, there is accumulating evidence that endocannabinoids (like anandamide) can influence reproduction, including fertilization, implantation, angiogenesis, embryo development and placental growth (Taylor et al., 2007; Habayeb et al., 2008a; Lewis et al., 2012; Solinas et al., 2012; Sun and Dey, 2012). One possible route to convey the effect of endocannabinoids is through the CB1 and CB2 receptors that are express by the human trophoblast and provide a direct target for cannabinoids (Kenney et al., 1999; Habayeb et al., 2008a; Habayeb et al., 2008b). However, not all endocannabinoids exert their effect through the classic cannabinoids receptors. For example CBD, lacks the central effects of cannabis and works through CB1 and CB2 independent model of action (Scuderi et al., 2009). Since our goal was to test the direct cannabinoid effect on trophoblast transporters BCRP and P-gp, and CBD showed the most potent inhibitory effect (among major cannabinoids) on these transporters (Holland et al., 2006; Zhu et al., 2006; Holland et al., 2007) it made CBD the ideal cannabinoid for the present study. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 10 BCRP and P-gp play a key role in transport of drugs and endogenous compounds in the human placenta, affecting the outcome of pregnancy (Robey et al., 2009; Myllynen et al., 2010). P-gp is expressed in the apical membrane of syncytiotrophoblast and probably is the main placental protective transporter during the first trimester. Its expression (both mRNA and protein) decreases with advancing gestation, being the highest during the first trimester (Gil et al., 2005; Mathias et al., 2005; Sun et al., 2006). BCRP plays an important role as a survival factor in BeWo cells as well as in the human placenta. It is thought to have a protective antiapoptotic role in the trophoblast, regulating their survival under low oxygen conditions (Krishnamurthy and Schuetz, 2006; Yeboah et al., 2006; Evseenko et al., 2007a; Evseenko et al., 2007b). The trophoblast expression of BCRP may change in different pregnancy complications. Indeed, placentas of women with preterm labor and intra-amniotic inflammation had a higher expression of this transporter than those of women with preterm labor without inflammation. In addition, the mRNA expression of BCRP correlated with that of IL-8, which also increased significantly in placentas of women with preterm labor and inflammation, suggesting that the transfer of drugs across the placenta may be altered in cases of preterm labor with inflammation (Mason et al., 2011 .( In the current study we have demonstrated for the first time that the exposure of trophoblast cell lines BeWo and Jar to CBD is associated with two distinct patterns of effects on the expression of the BCRP and P-gp transporters. The use of these cell lines, instead of primary trophoblast cultures, results from the fact that primary trophoblast may rapidly differentiate in culture, continuously changing their gene and protein expression (Evseenko et al., 2006). Comparison of these parameters in regard to CBD influences in such dynamic in vitro environment would be almost impossible. Moreover, BeWo and Jar cell lines, derived from human gestational choriocarcinoma, commonly used as an in vitro model for trophoblast toxicology studies. They offer a suitable model to study certain aspects of human trophoblast 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 11 physiology, without the aspect of inter-patient variability (Sullivan, 2004; Khare et al., 2006; Myren et al., 2007.( The first effect that was observed following chronic exposure is that CBD transcriptionally inhibited P-gp membrane expression (as mRNA levels of P-gp were also dramatically reduced following long-term exposure to CBD). The chronic inhibitory effect of CBD on P-gp protein was previously reported in drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) (Holland et al., 2006). The cell specificity of CBD was previously reported (Khare et al., 2006; Hu et al., 2011), yet our study is the first to detect this phenomenon in trophoblast cell lines. This observation may have clinical implications, in light of the role of P-gp as one of the key transport mechanisms for numerous drugs in the human placenta, especially during the venerable period of the first trimester in which all the fetal organs are formed. Thus, our results might be especially important to women who use cannabis on a regular basis during the first trimester and are treated with other drugs that are P-gp substrates . The expressional up-regulation of BCRP mRNA and protein in trophoblast cell lines following chronic exposure to CBD is the second novel effect reported in our study. This observation is similar to that reported in trophoblast of women with preterm labor and inflammation, suggesting that I) chronic exposure to CBD may elicit an inflammatory response in trophoblast cell lines (Mason et al., 2011); and II) that this phenomenon could be a direct compensational consequence of P-gp down-regulation, providing a working defense line to the developing embryo. Interestingly, compensation of such nature was already described in murine placentas (Hutson et al., 2010.( Of note, many of the drugs that are prescribed and considered safe to use during pregnancy are in fact P-gp or BCRP substrates, like Loratadine and H2 blockers (i.e. Ranitidine and Cimetidine) (Collett et al., 1999; Chen et al., 2003; Li et al., 2008; Schwarz et al., 2008; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 12 Dahan and Amidon, 2009; Gill et al., 2009; Matok et al., 2010). Our finding may have clinical implications, suggesting that the use of cannabis during gestation may alter drug transport through the trophoblast and lead to the absence of a functional placental barrier during the first trimester, leaving the developing embryo unprotected at this vulnerable period of pregnancy. Moreover, the trophoblast of pregnant women exposed to marijuana patients may exhibit some resistance to apoptotic and inflammatory processes due to the effect of CBD on BCRP expression. Conclusions: Following cannabis consumption, all the drugs that are P-gp substrates can potentially penetrate the human placental barrier at higher rates when combined with CBD, and therefore their safety under these conditions is to be questioned. Additionally, changes in placental BCRP expression profile might lead to altered transplacental transport of BCRP substrates, such as medications, naturally occurring carcinogens, hormonal precursors and apoptotic molecules, and influence pregnancy outcomes. methods : Cell culture and drug treatments: MCF7/P-gp, BeWo and Jar cells were cultured as previously described (Golan et al., 2009; Feinshtein et al., 2010). Briefly, 24h after seeding in 35 x 10 mm cell culture dishes (Corning), growth medium (DMEM for MCF7/P-gp, DMEM/F12 for BeWo and Jar cells) was replaced with fresh one containing CBD 10 or 15\u00b5M (initially dissolved in DMSO) or DMSO (0.08% or 0.12%, respectively, vehicle control). In order to achieve chronic exposure, treatment medium was refreshed every day for 24, 48 or 72h . Subcellular fractionation: Fractionation procedure was carried out as previously described (Golan et al., 2009), for the isolation of the membrane fraction. The membrane fraction was diluted in sample buffer 1:3 (10% v/v glycerol, 20% v/v SDS 20%, 5% v/v \u03b2-mercaptoethanol, 0.05% w/v bromophenol blue, pH 6.8), boiled for 5 min at 950C (for BCRP determination) or incubated for 30 min at 370C (for P-gp determination) and frozen at -800C until assayed. Aliquots were taken for protein determination using the Lowry assay. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 5 Fractions purity was verified using specific markers: Na+/K+ ATPase for membranes and NF/kB p65 for cytosol . valeria feinshtein1*, offer erez2, zvi ben-zvi1, tamar eshkoli2, boaz sheizaf2, eyal : sheiner2, mahmud huleihel3, gershon holcberg2. : 1 - Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. 2 - Department of Obstetrics and Gynecology, Soroka University Medical Center, School of medicine, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84101, Israel. 3 - The Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. *Corresponding author: Valeria Feinshtein M.Med.Sc. Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev . P.O. Box 653, Beer-Sheva, Israel, 84105. Tel.: +972 8 6477373; fax: +972 8 6479303; E-mail: shteiman@bgu.ac.il 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 2 immunocytochemistry and confocal microscopy : MCF7/P-gp, BeWo and Jar cells were seeded on cover slips and grown to 70% sub-confuency. Cells were then fixed in PFA for 15min and stained with mouse anti-human CD 243 (MDR-1) antibody, diluted in PBS, containing 3% BSA, for 1h at room temperature, followed by incubation with Alexa Fluor - 488 goat anti-mouse antibody in PBS containing 3% BSA, for 1/2h at room temperature. Cover-slips were washed 3 times and mounted onto glass slides with Dapi-containing fluorescent mounting medium (DapiFluoromount-6). Immunofluorescence was detected by an Olympus FV-1000 Spectral confocal laserscanning microscope with excitation at 488nm and emission at 520nm. Image analysis was performed using ImageJ v. 1.40C software. Immunoblotting Membrane fractions were thawed on the day of assay. Protein aliquots (60-100\u00b5g/lane) were taken for protein separation by SDS-PAGE as previously described (Golan et al., 2009). Semi-quantitative analysis was carried out using a computerized image analysis system (EZQuant-Gel 2.11, EZQuant Biology Software Solutions Ltd., Israel). Equal protein loading was ensured by normalization to Na+/K+ ATPase (for P-gp) or actin (for BCRP .( RNA extraction, reverse transcription, Real-Time Polymerase Chain Reaction (qPCR( Isolation and purification of total RNA from BeWo and Jar cells was carried out using EZ-RNA Kit (Biological Industries, Israel) according to manufacturer's instructions. 1\u00b5g of total RNA was used for reverse transcription using High Capacity cDNA RT kit, in 20\u00b5l 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 6 reaction volume. BCRP and P-gp mRNA was measured by qRT-PCR, as indicated in the manufacturer protocol (Applied Biosystems, Foster City, CA), and performed by the Applied Biosystems Real Time PCR system (7500 system), using TaqMan probes and primers for human BCRP, P-gp and actin (Applied Biosystems, Foster City, CA). The cycling conditions for all primers were as follows: hold for 10 min at 95\u00b0C, followed by 40 cycles consisting of two steps, 15 s at 95\u00b0C (denaturing), and 1 min at 60\u00b0C (annealing-extension). The threshold cycle, which correlates inversely with the mRNA levels of target, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level. P-gp and BCRP mRNA levels were normalized to actin mRNA in the same samples. Results were analyzed by the 2(-\u0394\u0394C(T)) method, demonstrating the relative changes in gene expression from real-time quantitative PCR experiments, using 7500 Software v2.0.4 (Applied Biosystems, Foster City, CA.( Statistical analysis: All statistics and graphs were carried out using GraphPad Prism5 software. One-way ANOVA followed by appropriate Bonferroni corrections was used. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t 7 72h time points. data is displayed in means \u00b1 s.d. of at least three independent experiments of each : concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test, **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated. PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t Figure 3 Chronic exposure of Jar cells to CBD: changes in membrane BCRP and P-gp expressed levels. Chronic exposure of Jar cells to CBD: changes in membrane BCRP and P-gp expressed levels. Changes in BCRP - CBD concentration-dependent (B) and time-dependent (A). Changes in P-gp (C). Data is displayed in means \u00b1 s.d. of at least three independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test (Student's t test for (C)), *p<0.05, ***p<0.0001 compared to control or comparison between groups as indicated. PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t Figure 4 Chronic exposure of MCF/P-gp cells to CBD Chronic exposure of MCF/P-gp cells to CBD: changes in membrane P-gp (A) and BCRP (B) expressed levels. Protein levels are given as percent of control levels. Data is displayed in means \u00b1 s.d. of at least three independent experiments of each concentration or time point. One representative blot is presented for each experimental group. One-Way ANOVA, followed by Bonferroni's multiple comparison test. **p<0.01, ***p<0.0001 compared to control or comparison between groups as indicated. PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t Figure 5 Chronic exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. Chronic exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. (A), (C) changes in BCRP mRNA in BeWo and Jar cells (respectively). (B), (D) changes in P-gp mRNA in BeWo and Jar cells (respectively). Values are given as fold of change compared to control . Data is displayed as means \u00b1 s.d. of at least three independent experiments for each time point. One-Way ANOVA, followed by Bonferroni's multiple comparison test. *p<0.05, **p<0.01, ***p<0.0001 compared to control. PeerJ reviewing PDF | (v2013:05:527:0:0:NEW 27 May 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/154/reviews/",
    "review_1": "John Bruno \u00b7 Aug 18, 2013 \u00b7 Academic Editor\nACCEPT\nI think the paper is good shape for production.",
    "review_2": "John Bruno \u00b7 Aug 14, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nFirst. I want to thank you for submitting to PeerJ and also to apologize for the too long review time. I had ten people decline my invitation to review. Probably a record, but does seem to be getting harder and harder to find reviewers.\n\nBoth reviewers were supportive and only asked for very minor revisions. Can you do this quickly and resubmit? When I get it back, I will not send it out again and will make a final decision within 24 hours.",
    "review_3": "Elvira Poloczanska \u00b7 Aug 10, 2013\nBasic reporting\nI agree with the authors that this study represents an advancement of our understanding of physical processes operating on littoral shores. The authors present and conceptual model and modelling framework that can be applied across a range or temporal and spatial scales. The background leading to this study is presented succinctly and demonstrates the foundations for this body of work.\nExperimental design\nNo comments\nValidity of the findings\nSee above\nAdditional comments\nParagraph starting line 52: The authors may wish to consider expanding the discussion re community composition and wave exposure eg see Burrows et al 2008 Wave exposure indices from digital coastlines and the prediction of rocky shore community structure. Marine Ecology Progress Series 353: 1-12 and Thomas 1986 A physically derived exposure index for marine shorelines. Ophelia 25:1-13.\n\nLine 79-87: I suggest bringing in a sentance to highlight that tidal regimes are also differ regionally,eg semi-diurnal, diurnal or semi-mixed,\n\nLine 112: I think you are referring to Fig 3a not Fig 1.\n\nLine: 159: incorrect spelling \u201cco-domintated\u201d\nCite this review as\nPoloczanska E (2013) Peer Review #1 of \"Between tide and wave marks: a unifying model of physical zonation on littoral shores (v0.1)\". PeerJ https://doi.org/10.7287/peerj.154v0.1/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Aug 8, 2013\nBasic reporting\nThis paper presents a novel model that explores the interaction between tidal and wave forces in generating intertidal zonation patterns. I have heard the senior author speak at national meetings about this model and seen a couple versions of this paper previously.\nIt should have been published years (6 or so) ago when it was first presented, but it unreasonably offended established rocky intertidal ecologists who were too set in their ways to accept fresh thinking about the drivers of intertidal zonation. Frankly, this should have been an ecology paper and in textbooks. It improves upon all the earlier models of Coleman, Doty and the Stephenson\u2019s.\n\nThe disparity between the intertidal mafia and a bright ambitious ecologist working alone in Hawaii w/o contact with high profile intertidal ecologists was that most established intertidal ecologists worked in habitats with large tidal ranges, ranges greater than local wave amplitudes. Bird was working in Hawaii where the tidal amplitude far exceeded the tidal amplitude. He did outstanding community assembly experimental shoreline ecology on his system, but since he was working alone in a system with very different parameters his work met resistance from the intertidal establishment on all his experimental work. This model incorporating these disparities in a general model is an outstanding example of originality coming out of a novel system that can teach us a general lesson. That his work was blackballed by established intertidal ecologist is a sad statement about how established dogma and their protagonists can hold back advancement based on myopic ideas.\n\nThis paper is already in perfect condition after numerous submissions, improvements and rewrites. Unfortunately, Bird has moved on to molecular evolution work where he is excelling, leaving the pettiness of rocky intertidal ecologists behind. He could have been an exceptional experimental community ecologist with better mentoring and more open-minded rocky intertidal researchers.\nExperimental design\nN/A\nValidity of the findings\nN/A\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Between tide and wave marks: a unifying model of physical zonation on littoral shores (v0.1)\". PeerJ https://doi.org/10.7287/peerj.154v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/154v0.2/submission",
    "pdf_2": "https://peerj.com/articles/154v0.1/submission",
    "all_reviews": "Review 1: John Bruno \u00b7 Aug 18, 2013 \u00b7 Academic Editor\nACCEPT\nI think the paper is good shape for production.\nReview 2: John Bruno \u00b7 Aug 14, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nFirst. I want to thank you for submitting to PeerJ and also to apologize for the too long review time. I had ten people decline my invitation to review. Probably a record, but does seem to be getting harder and harder to find reviewers.\n\nBoth reviewers were supportive and only asked for very minor revisions. Can you do this quickly and resubmit? When I get it back, I will not send it out again and will make a final decision within 24 hours.\nReview 3: Elvira Poloczanska \u00b7 Aug 10, 2013\nBasic reporting\nI agree with the authors that this study represents an advancement of our understanding of physical processes operating on littoral shores. The authors present and conceptual model and modelling framework that can be applied across a range or temporal and spatial scales. The background leading to this study is presented succinctly and demonstrates the foundations for this body of work.\nExperimental design\nNo comments\nValidity of the findings\nSee above\nAdditional comments\nParagraph starting line 52: The authors may wish to consider expanding the discussion re community composition and wave exposure eg see Burrows et al 2008 Wave exposure indices from digital coastlines and the prediction of rocky shore community structure. Marine Ecology Progress Series 353: 1-12 and Thomas 1986 A physically derived exposure index for marine shorelines. Ophelia 25:1-13.\n\nLine 79-87: I suggest bringing in a sentance to highlight that tidal regimes are also differ regionally,eg semi-diurnal, diurnal or semi-mixed,\n\nLine 112: I think you are referring to Fig 3a not Fig 1.\n\nLine: 159: incorrect spelling \u201cco-domintated\u201d\nCite this review as\nPoloczanska E (2013) Peer Review #1 of \"Between tide and wave marks: a unifying model of physical zonation on littoral shores (v0.1)\". PeerJ https://doi.org/10.7287/peerj.154v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Aug 8, 2013\nBasic reporting\nThis paper presents a novel model that explores the interaction between tidal and wave forces in generating intertidal zonation patterns. I have heard the senior author speak at national meetings about this model and seen a couple versions of this paper previously.\nIt should have been published years (6 or so) ago when it was first presented, but it unreasonably offended established rocky intertidal ecologists who were too set in their ways to accept fresh thinking about the drivers of intertidal zonation. Frankly, this should have been an ecology paper and in textbooks. It improves upon all the earlier models of Coleman, Doty and the Stephenson\u2019s.\n\nThe disparity between the intertidal mafia and a bright ambitious ecologist working alone in Hawaii w/o contact with high profile intertidal ecologists was that most established intertidal ecologists worked in habitats with large tidal ranges, ranges greater than local wave amplitudes. Bird was working in Hawaii where the tidal amplitude far exceeded the tidal amplitude. He did outstanding community assembly experimental shoreline ecology on his system, but since he was working alone in a system with very different parameters his work met resistance from the intertidal establishment on all his experimental work. This model incorporating these disparities in a general model is an outstanding example of originality coming out of a novel system that can teach us a general lesson. That his work was blackballed by established intertidal ecologist is a sad statement about how established dogma and their protagonists can hold back advancement based on myopic ideas.\n\nThis paper is already in perfect condition after numerous submissions, improvements and rewrites. Unfortunately, Bird has moved on to molecular evolution work where he is excelling, leaving the pettiness of rocky intertidal ecologists behind. He could have been an exceptional experimental community ecologist with better mentoring and more open-minded rocky intertidal researchers.\nExperimental design\nN/A\nValidity of the findings\nN/A\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Between tide and wave marks: a unifying model of physical zonation on littoral shores (v0.1)\". PeerJ https://doi.org/10.7287/peerj.154v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: I think the paper is good shape for production.\\n\\nReview 2: Both reviewers were supportive and only asked for very minor revisions. Can you do this quickly and resubmit? \\n\\nReview 3: The authors may wish to consider expanding the discussion re community composition and wave exposure. I suggest bringing in a sentence to highlight that tidal regimes also differ regionally. I think you are referring to Fig 3a not Fig 1. Incorrect spelling \u201cco-domintated\u201d.\\n\\nReview 4: This paper presents a novel model that explores the interaction between tidal and wave forces in generating intertidal zonation patterns. It should have been published years ago when it was first presented, but it unreasonably offended established rocky intertidal ecologists. The disparity between the intertidal mafia and a bright ambitious ecologist working alone in Hawaii was significant. This model incorporating these disparities in a general model is an outstanding example of originality. This paper is already in perfect condition after numerous submissions, improvements, and rewrites. \\n\\nReview 5: nan\\n\\nReview 6: ']"
}