| "text": "This is an academic paper. This paper has corpus identifier PMC2527472\nAUTHORS: Yuki Ito, Tamie Nakajima\n\nABSTRACT:\nDi(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer and a potentially nongenotoxic carcinogen. Its mechanism had been earlier proposed based on peroxisome proliferator-activated receptor α (PPARα) because metabolites of DEHP are agonists. However, recent evidence also suggests the involvement of non-PPARα multiple pathway in DEHP-induced carcinogenesis. Since there are differences in the function and constitutive expression of PPARα among rodents and humans, species differences are also thought to exist in the carcinogenesis. However, species differences were also seen in the lipase activity involved in the first step of the DEHP metabolism, which should be considered in DEHP-induced carcinogenesis. Taken together, it is very difficult to extrapolate the results from rodents to humans in the case of DEHP carcinogenicity. However, PPARα-null mice or mice with human PPARα gene have been developed, which may lend support to make such a difficult extrapolation. Overall, further mechanical study on DEHP-induced carcinogenicity is warranted using these mice.\n\nBODY:\n1. INTRODUCTIONDi(2-ethylhexyl)phthalate\n(DEHP) a plasticizer around the world, suggesting that many\npeople come across this chemical every day. Animal studies showed that this\nchemical is a nongenotoxic carcinogen. Metabolites of DEHP, mono- and\ndicarboxylic acids, transactivate peroxizome proliferator-activated receptor α (PPARα),\nwhich has been thought to result in nongenotoxic carcinogenesis [1, 2]. However, the latest studies also\nshowed the involvement of non-PPARα pathways; multiple pathways might be\ninvolved in the pathway of DEHP-induced carcinogenicity [3]. There are species differences in\nthe functional activation or constitutive expression of rodent and human PPARα,\nand that in humans is thought to be less active and expressive than those of\nrodents. Recently, inflammation-related carcinogenesis has drawn attention [4, 5]. PPARα is involved not only in\nthe induction of target genes such as β-oxidation enzymes of fatty acids but also in anti-inflammation\nsignaling [6, 7], suggesting that PPARα also may protect\nagainst carcinogenesis. Species differences in lipase activity (DEHP-metabolizing\nenzyme) among mice, rats, and marmosets have been also reported recently [8], suggesting that this kinetic\ndifference should be considered in the species differences in DEHP-induced\ncarcinogenesis. In this review, we focused on DEHP-induced hepatic\ncarcinogenesis in relation to PPARα-dependent and PPARα-independent pathways, and discussed\nthe science policy.2. PPARsPPARs are\ninvolved in a member of the nuclear hormone receptor superfamily, and consist\nof three subunits: PPARα, PPARβ/δ, and PPARγ [9]. These three isoforms have been\nidentified at the organ-specific level. In the respective organ, PPARs function\nas transcription factors through the classic ligand-dependent nuclear hormone\nreceptor mechanism. Upon binding to their ligands, PPARs undergo conformational\nchanges that allow corepressor release [10]. The PPAR-ligand complex binds to\ndirect repeat 1 elements or peroxisome proliferator response elements (PPREs),\nusually located upstream of the target genes, which results in the induction of\nfatty acid transport and metabolism, glucose metabolism, and also elicitation\nof anti-inflammatory effects [6, 11].As one of the three isoforms, PPARα is mainly expressed in\norgans that are critical in fatty acid catabolism, such as liver, heart, and\nkidney [7]. Thus, this nuclear receptor is\nprimarily involved in the regulation of fatty acid metabolism. In addition to\nthis function, PPARα\nalso has various functions including the promotion of gluconeogenesis,\nlipogenesis, ketogenesis, and anti-inflammatory effects [6].3. PPARα LIGANDSThe ligands of PPARα represent a diverse group of\nchemicals including not only endogenous ligands but also exogenous synthetic\nligands with a high likelihood of clinical, occupational, and environmental\nexposure of humans to chemicals [1, 12]. The primary endogenous ligands\nare fatty acids, mainly the 18–20 carbon polyunsaturated fatty acids and\neicosanoids [7, 13–17]. As exogenous ligands, fibrates\nand thiazolidinediones are involved. Additionally, the general population is\nexposed to environmental chemicals such as plasticizers (e.g., phthalates),\nsolvents (e.g., tetrachloroethylene and trichloroethylene), perfluorooctanoic\nacid and herbicides (e.g., 2, 4-dichlorophenoxyacetic acid, diclofop-methyl,\nhaloxyfop, lactofen, and oxidiazon).Of these ligands, the toxicity of DEHP is well\nestablished in relation to PPARα. This chemical is used as a\nplasticizer to improve the plasticity and elasticity of polyvinyl chloride\nproducts that have become ubiquitous in our daily living. These products are\nwidely used in building materials, wallpaper and flooring, wire covering, vinyl\nsheeting for agriculture, food packages, and medical devices such as\nintravenous and hemodialysis tubing and blood bags. The recent production of\nDEHP in Japan\nhas approached 14 000 tons per year, which accounts for about 54% of all\nplasticizers used [11]. It is noted that mono- and dicarboxylic acid\nmetabolites of DEHP, not DEHP itself, act as ligands for PPARα\n[18] and have potentially adverse\neffects on liver, kidney, heart, and reproductive organs though monocarboxylic\nacid, mono(2-ethylhexyl) phthalate (MEHP), also binds to PPARγ [18].4. SPECIES DIFFERENCES IN PPARα\nSince there are\nspecies differences in the toxicity of PPARα agonists, the expression levels or\nfunctions of the receptor are thought to be different among species. Several\nexplanations for the species differences in response to the ligands have been\nsuggested [19, 20]. One of the major factors was considered\nto be due to differences in the levels of PPARα expression [21, 22] although other possibilities include\ndifferences in ligand affinity between rodent and human PPARα, differences in\ncellular context of PPARα expression, and those in PPRE sequences found upstream\nof critical target genes [23, 24]. Indeed, PPARα expression in\nhumans is about 1/10 times less than that in rodents [25]. In addition, micro-RNA\nexpression regulated by PPARα has been recently reported to be changed in\nwild-type mice, but not in mice with human PPARα gene [26]; Wy-14,643 inhibited a micro-RNA\nlet-7C which is involved in suppression of tumorigenesis in wild-type mice, but\nneither in PPARα-null mice nor in mice with human PPARα gene. Mice\nwith human PPARα gene are resistant to hepatocellular proliferation though they\nrespond to Wy-14,643 in β-oxidation and serum triglycerides [27]. These results suggest that the function\nof the PPARα signaling in liver proliferation and tumorigenesis by the chemical\nexposure is not always similar in mice and humans.In regard to the species differences in\nthe PPREs, the lack of acyl CoA oxidase (ACO) induction in studies on liver\nbiopsies from humans treated with hypolipidemic drugs or primary human\nhepatocytes treated with Wy-14,643 may be attributable to an inactive\nfunctional PPRE since the sequence of a PPRE for the ACO gene from a small\nnumber of human liver biopsy samples was found to be different from that of the\nrats [28]. However, Reddy remarked at a\npanel discussion that, although the sequence of ACO gene promoter in the mouse\nwas also different from that in the rat, both rodents are responsive to some\nperoxisome proliferators in ACO induction [20]. In addition, differences in the\nability of rodents and human PPAR to recognize and bind PPRE are unlikely since\nthe DNA binding domains of the human and rodent PPARα are 100% homologous [29, 30]. Though characterized from only a\nlimited number of individuals, the prevalence in the population of defective\nPPAR alleles cannot be determined at this point [31]. The species difference in the\nsequence of PPRE may not be involved in the difference in response to ligands\nbetween rodents and humans.In addition to the lower expression\nlevels of PPARα in human, there was a truncated, inactive form of PPARα in\nhuman liver, suggesting that the expression of full-length functional PPARα was\nvery low. These inactive forms of PPARα may be insufficient to bind PPRE\nbecause PPREs may be occupied in vivo by other nuclear receptors that bind to\nsimilar sequences, thus affecting responsiveness to ligands [25].5. SPECIES DIFFERENCES IN DEHP METABOLISMIn addition to\nthe species differences in PPARα functions or expression levels, we should also\nbe mindful of the importance of those in the metabolism of DEHP between rodents\nand humans. DEHP absorbed in the body is first metabolized by the catalytic\naction of lipase to produce MEHP and 2-ethylhexanol (2-EH) [32]. Some MEHP is then conjugated\nwith UDP-glucuronide by UDP-glucuronosyltransferase (UGT) and excreted in the\nurine. The remaining MEHP is excreted directly in the urine or is oxidized by\ncytochrome P450 4A, then further oxidized by alcohol dehydrogenase (ADH) or\naldehyde dehydrogenase (ALDH) to dicarboxylic acid or ketones. 2-EH is metabolized\nmainly to carboxylic acid (mainly 2-ethylhexanoic acid (2HEA)) via 2-ethylhexanal\nby catalytic action of ADH and ALDH. Thus, lipase may be an essential enzyme to\nregulate the DEHP metabolism; knowing the species difference in the lipase\nactivity may be an important tool to clarify the species difference in\nmetabolism.Recently,\nthe activities of lipase, UGT, ADH, and ALDH for DEHP metabolism in several\norgans were measured and compared among mice, rats, and marmosets [8]. Marmosets were used as a\nreference to human. Clear-cut species differences were seen in the activities\nof the four enzymes involved in the DEHP metabolism among mice, rats, and\nmarmosets. The most prominent difference was observed in the lipase activity\nwith an almost 148- to 357-fold difference between the highest activity in mice\nand the lowest in marmosets (Figure 1). These differences were comparable to\nthose in the kinetic parameter, Vmax. These results suggest that the constitutive levels of lipase were\ngreater in the mice and rats than in marmosets. Indeed, lipase-mRNA\nlevels in livers from mice or rats were much higher than those in marmoset (Figure\n2). Thus, concentrations of MEHPs (ligands to PPARα) in the body were\nhigher in mice or rats than in marmosets when the same dose of DEHP was\nadministered [33].Besides species differences in the\nconstitutive levels of lipase, Km values of DEHP for lipase of marmosets\nwere much higher than in rats or mice, suggesting the species differences in\nthe DEHP affinity for lipase; the affinity of DEHP for lipase in the marmosets\nmay be lower than that of mice or rats. The affinity in human may be even lower\nthan that in primates; cumulative 14C excretion in urine of African green\nmonkey following bolus injection of 14C-DEHP leached into autologous\nplasma occurred earlier than in human [34].6. MECHANISM OF DEHP-INDUCED CANCERDEHP\ncauses tumors, especially in liver when chronically administered to rats and\nmice [35–39],\nsimilar to the other peroxisome proliferators such as Wy-14643. Table 1 shows that DEHP induces hepatic\ntumors in mice and rats. From the viewpoint of percentage in feed, the lowest-observed\neffect-level (LOEL) of DEHP carcinogenicity in the rat was 0.6%, and the no-observed\neffect-level (NOEL) was 0.1% [2]. In the mouse, the corresponding values may\nbe 0.05% for LOEL and 0.01% for NOEL because the study in which male mice were exposed\nto 0.05% DEHP for 78 weeks exhibited a significant increase in the hepatic\ntumor incidence rate compared with controls, but not when exposed to 0.01% DEHP\n[40].DEHP also has potential for carcinogenesis in\nother organs; pancreatic acinar cell adenoma and mononuclear cell leukemia\nincidences were significantly increased in male F344 rat but not in F344 female\nrat and B6C3F1 mouse of both sexes after DEHP exposure [35, 36, 44]. The reason why these cancers are not\nobserved in female rat has not been identified.Chronic\ntreatment with PPARα agonist results in an increased incidence of liver tumors\nwhich were thought to have occurred through a PPARα-mediated mechanism as\nrevealed by the resistance of PPARα-null mice to liver cancer induced by\nWy-14,643 exposure for 11 months [46]. All the\nwild-type mice fed with 0.1% Wy-14643 diet for 11 months had multiple\nhepatocellular neoplasms, including adenomas and carcinomas, while thePPARα-null\nmice fed with the 0.1% Wy-14643 diet for the same duration were unaffected.\nWard et al. [47] reported that\nexposure for only six months to 12 000 ppm DEHP caused induction of peroxisomal\nenzymes, liver enlargement, and histopathological increases in eosinophil\ncounts and peroxisomes in the cytoplasm of wild-type mice, while there were no\nsuch toxic findings in the liver of PPARα-null\nmice. Thus, DEHP-derived carcinogenicity was thought to be mediated by PPARα,\nsimilar to Wy-14,643, and DEHP was considered to cause primarily\nPPARα-dependent carcinogenicity in rodents, but it is considered to be\nrelatively safe in humans, similar to other ligands [2]. However, Ward\net al. [47] could not directly\nobserve DEHP-derived tumors in the wild-type mice, because exposure to DEHP for 6 months may\nnot be sufficient to induce hepatic tumors, as suggested by Marsman et al. [48]; they reported that DEHP\ntumorigenesis required longer exposure periods than Wy-14,643.\nIt is doubtful whether DEHP definitively induces hepatic tumors via PPARα.As mentioned\nabove, the following simple mechanism has been proposed for the DEHP-induced\nhepatocarcinogenesis; when DEHP was administered to rats and mice, the chemical\ncaused an increase in cell proliferation and peroxisome proliferation [49]. The latter\nis accompanied by an increase in both peroxisomal and mitochondrial fatty acid\nmetabolizing enzymes such as ACO. As a byproduct of fatty acid oxidation,\nenzymes involved with β-oxidation generate H2O2,\nresulting in elevated oxidative stress. DEHP also causes an increase in proinflammatory\ncytokines and inhibition of apoptosis [2, 24].DEHP-induced\nliver carcinogenesis in rodents, however, appears to involve more complex\npathways as described in the following events whereby various combinations of\nthe molecular signals and multiple pathways may be involved [3]. DEHP is\nmetabolized to bioactive metabolites which are absorbed and distributed\nthroughout the body; they might induce PPARα-independent activation of\nmacrophages and production of oxidants, and also activate PPARα and sustained\ninduction of target genes. The inductions lead to enlargement of hepatocellular\norganelles, an increase in cell proliferation, a decrease in apoptosis, sustained\nhepatomegaly, chronic low-level oxidative stress and accumulation of DNA damage,\nand selective clonal expansion of the initiated cells. Finally, preneoplastic\nnodules might be induced and might result in adenomas and carcinoma.Peraza et al.\n[10] also suggest that PPARα\nis the only receptor in PPARs that is known to mediate carcinogenesis, while\nthe prevailing evidence suggests that PPARβ, PPARγ, and their ligands appear to\nbe tumor modifiers that inhibit carcinogenesis, albeit there is still\ncontroversy in the field. Melnick [50] also\naddressed non-PPARα mechanisms for DEHP-induced carcinogenicity as follows. (1)\nPeroxisome\nproliferator-induced tumorigenesis is related to the genes involved in cellular\nproliferations of, for example, p38 mitogen-activated protein kinase, which is\nnot involved in peroxisome proliferations [51]. (2) DEHP and other peroxisome\nproliferators stimulated growth regulatory pathways such as immediate early\ngenes for carcinogenesis (c-jun, c-fos, junB, egr-1), mitogen-activated protein\nkinase, extracellular signal-regulated kinase, and phosphorylation of p38,\nwhich were dissociated from PPARα activation in rat\nprimary cultures [52–54]. These findings also support the\nview that peroxisome proliferators, including DEHP, may have the potential for\ntumorigenesis via non-PPARα signal pathways.In recent\nyears, an inflammation-associated model of cancers has been given attention [4, 5].\nPPARα exerts anti-inflammation effects by repressing nuclear factor kappa B\n(NFκB) [55], which\ninhibits inflammation signaling and subsequent cancer [4].Ito et al. [45]\nproposed possibility of DEHP tumorigenesis via a non-PPARα pathway using PPARα-null mice. They compared DEHP-induced\ntumorigenesis in wild-type and PPARα-null mice treated\nfor 22 months with diets containing 0, 0.01, or 0.05% DEHP. Surprisingly, the\nincidence of liver tumors was higher in PPARα-null\nmice exposed to 0.05% DEHP (25.8%) than in similarly exposed wild-type mice (10%), while the incidence was 0% in wild-type miceand 4% in PPARα-null\nmice without DEHP exposure. The levels of 8-hydroxydeoxyguanosine increased\ndose-dependently in mice of both genotypes, but the degree of increase was\nhigher in PPARα-null mice than in\nwild-type mice. NFκB\nlevels also significantly increased in a dose-dependent manner in PPARα-null mice. The proto-oncogene\nc-jun-mRNA was induced, while c-fos-mRNA tended to be induced only in PPARα-null mice fed with 0.05%\nDEHP-containing diet. These results suggest that chronic low-level oxidative\nstress induced by DEHP exposure may lead to the induction of inflammation\nand/or the expression of proto-oncogenes, resulting in a high incidence of\ntumorigenesis in PPARα-null mice. Moderate activated PPARα might protect from p65/p50 NFκB inflammatory pathway caused by\nchronic DEHP exposure in wild-type mice. Although cross-talk of PPARγ,\nbut not PPARα, with cyclooxygenase 2 (Cox-2), which also was related\nwith inflammation-induced hepatocellular carcinoma, has been suggested [56], there was neither induction of\nCox-2 nor PPARγ in both genotyped mice of that study (data\nnot shown).Additionally, we compared the mechanisms\nof tumorigenesis between wild-type mice and PPARα-null\nmice using hepatocellular adenoma tissues of both genotyped mice [57]. The microarray profiles showed\nthat the up- or downregulated genes were quite different between hepatocellular\nadenoma tissues of wild-type mice and PPARα-null\nmice exposed to DEHP, suggesting that their tumorigenesis mechanisms might be\ndifferent. Interestingly, the gene expressions of apoptotic peptidase\nactivating factor 1 and DNA-damage-inducible 45α (Gadd45α) were increased in\nthe hepatocellular adenoma tissues of wild-type mice exposed to DEHP, whereas\nthey were unchanged in corresponding tissues of PPARα-null mice. On the other hand, the expressions of cyclin B2\nand myeloid cell leukemia sequence 1 were increased only in the hepatocellular\nadenoma tissues of PPARα-null mice.\nTaken together, DEHP may induce hepatocellular adenomas, partly via suppression\nof G2/M arrest regulated by Gadd45α and caspase 3-dependent apoptosis in PPARα-null mice. However, these genes\nmay not be involved in tumorigenesis in wild-type mice. In contrast, the\nexpression level of Met was notably increased in the liver adenoma tissue of\nwild-type mice, which may suggest the involvement of Met in DEHP-induced\ntumorigenesis in wild-type mice. However, we could not\ndetermine whether DEHP promoted the spontaneous liver tumor in PPARα-null\nmice because spontaneous hepatocellular tumors are known to occur in these mice\nat 24 months of age [58], while\nwe observed DEHP-induced tumorigenesis at 22 months of age. To clarify this,\ngene expression profiles of liver tumors in the control group must be analyzed.Taken together,\nthe mechanisms of DEHP-induced carcinogenesis do not consist of only a simple\npathway such as PPARα-mediated peroxisome proliferation as mentioned by Rusyn et\nal. [3].\nPPARα-independent pathways may also exist and, by contrast, activated PPARα may\nprotect against DEHP-induced carcinogenesis. The valance of the production of\noxidative stress via the transactivation of PPARα and subsequent DNA damages\nversus the effective exertion of anti-inflammation by activating the receptor may\ndetermine the incidence of DEHP-induced tumors.7. FUTURE INVESTIGATIONSTo determine the mechanism of species\ndifference in response to peroxisome proliferators, a mouse line with human PPARα\nwas produced and designated hPPARα\nTetOff [27]. This mouse line expresses the\nhuman receptor in liver in a PPARα-null\nbackground by placing the hPPARα cDNA under control of the Tet-Off system of\ndoxycycline control with the liver-specific LAP1 (C/EBPβ) promoter.\nInterestingly, the hPPARα\nTetOff mice express the human PPARα protein\nat levels comparable to those\nexpressed in wild-type mice; so we should not need to consider the species\ndifferences in the expression of PPARα between mice and humans. Treatment of\nthis mouse line with Wy-14,643 revealed induction of genes' encoding\nperoxisomal lipid-metabolizing enzymes, including ACO, bifunctional enzyme and peroxisomal\nthiolase, and the fatty acid transporter CD36 at a level comparable to that in\nwild-type mice, expressing native mouse PPARα. This suggested that human PPARα\nis functionally active. Upon treatment with Wy-14,643, hPPARα\nTetOff mice also had lower levels of fasting serum total triglycerides similar to\nwild-type mice. However, hPPARα\nTetOff mice did not show any\nsignificant hepatocellular proliferation, nor did they have an induction of\ncell cycle control genes, in contrast to Wy-14,643-treated wild-type mice where\na significant increase in mRNAs encoding PCNA, cMYC, cJUN, CDK1, CDK4, and\nseveral cyclins was found after treatment with Wy-14,643. hPPARα\nTetOff mice were also found to be resistant to Wy-14,643-induced hepatocarcinogenesis\nafter 11 months of Wy-14,643 feeding in contrast to a 100% incidence in the\nwild-type mouse group [59].Another transgenic mouse line with human PPARα\nwas generated that has the complete human PPARα gene on a P1 phageartificial chromosome (PAC) genomic clone, introduced onto the mouse PPARα-null background [60]. This new line, designated hPPARα\nPAC,\nexpresses human PPARα not only in liver but also in kidney and\nheart. hPPARα\nPAC mice exhibited responses similar to wild-type mice\nwhen treated with fenofibrate lowering of serum triglycerides and induction of\nPPARα target genes' encoding enzymes involved in fatty acid metabolism.\nTreatment of hPPARα\nPAC mice with fenofibrate did not cause\nsignificant hepatomegaly and hepatocyte proliferation similar to hPPARα\nTetOff mice, suggesting that the resistance to the hepatocellular proliferation found\nin the hPPARα\nTetOff mice is not due to lack of expression of the\nreceptor in tissues other than liver.Until now, there are no reports concerning the\ninteraction between DEHP and hPPARα\nTetOff or hPPARα\nPAC.\nRecently, we have compared the transactivation of mouse and human PPARα by DEHP treatments\nusing wild-type and hPPARα\nTetOff mice (unpublished observation). A relatively\nhigh dose of DEHP (5 mmol/kg for 2 weeks) clearly activated PPARα in liver of\nboth genotyped mice, but the activation was very little in hPPARα\nTetOff mice from the standpoint of the target gene expression as well as triglyceride\nlevels in plasma and liver. Human PPARα response to DEHP may be weak when sufficient human PPARα is expressed in the\nhuman liver. Thus, the use of the hPPARα\nTetOff mouse model is a very\nvaluable means to solve the species differences in the toxicity of peroxisome\nproliferators. The results from the typical peroxisome proliferator (Wy-14643)\nmay not always be similar to those of DEHP; a study of each case is needed\nusing hPPARα\nTetOff mouse model.8. PROPOSED SCIENCE POLICY STATEMENTSThe International Agency for Research on Cancer downgraded the level\nof potential health risks of DEHP from 2b (possibly carcinogenic to humans) to 3 (not classifiable as to carcinogenicity to humans) in 2000 [61]. In\nthis report, DEHP carcinogenesis via PPARα was considered not to be relevant to\nhumans because peroxisome proliferation had not been documented either in human\nhepatocyte cultures exposed to DEHP or in the liver of nonhuman primates. This\ndecision has been variously argued by several scientists in the literature [50, 62, 63]. In contrast, the Japan Society\nfor Occupational Health has maintained the 2B class of DEHP carcinogenicity\nbecause of the obvious rodent carcinogenicity [64].Although the US Environmental\nProtection Agency (EPA) had classified the risk for DEHP carcinogenicity as B2 (probable human carcinogen) in 1993, recently, the expert panel of EPA report has provided\nthe current scientific understanding of the mode(s) of action of PPARα agonist-induced\ntumors observed in rodent bioassays that are associated with PPARα agonisms: liver\ntumors in rats and mice as well as Leydig cell and pancreatic acinar cell\ntumors in rats—all of which represent\nlimited evidence [65]. Since the key events for the mode of action, which have\nbeen causally related to liver tumor formation, include the activation of PPARα, perturbation of cell\nproliferation and apoptosis, selective clonal expansion, and the\nPPARα-related key events included in the expression of peroxisomal genes (e.g., palmitoyl CoA oxidase and acyl CoA oxidase) and\nperoxisome proliferation (i.e., an increase in\nthe number and size of peroxisomes) are reliable markers. Additionally, the\nevidence obtained from the findings that PPARa agonists did not activate the\nreceptor in human cell culture or biopsy samples, and from epidemiological\nstudies, shows that humans are apparently refractory to the effects of a PPARα\nagonist. However, the EPA maintained the DEHP carcinogenicity criterion.In 2004, with regard to preclinical and clinical safety assessments\nfor PPAR agonists, the Food and Drug Administration recommended that, due to the prevalence of positive tumor findings of PPAR\nagonists, two-year carcinogenicity studies on mice and rats are required [66].Although IARC changed the criterion for DEHP\ncarcinogenicity, other agencies did not because DEHP is a potential rodent\ncarcinogen of liver and the precise mechanism has not been yet understood,\nthough DEHP is a potentially hepatic carcinogen in rodents.9. CONCLUSIONSAs mentioned above, some studies suggest\nthe possibility of DEHP tumorigenesis via a non-PPARα pathway although DEHP also\nexerts adverse effects via PPARα-dependent pathway. 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