Peroxisome proliferators: response - Correspondence

The comments from Roberts et al. regarding my commentary (1) on whether human and mechanistic data provide a sufficient rationale to dismiss DEHP cancer risks because of species differences in peroxisome proliferation reveal both agreements and disagreements on this issue. We agree that the animal carcinogenicity data are irrefutable and, as written by Roberts et al., we agree that "the hepatocarcinogenicity [of peroxisome proliferators (PPs)] is unlikely to be caused by peroxisome proliferation per se." In addition, we agree that the mechanisms of carcinogenic action of DEHP and other PPs remain to be determined.

Where we differ is on our views of what constitutes sufficient evidence to dismiss positive animal cancer data as being indicative of possible human cancer risk, as well as on interpretations of available data. From a public health perspective, it is important to reexamine the basis on which Roberts et al. conclude that rodent liver carcinogenesis induced by PPs "is not relevant to humans" while acknowledging that the mechanisms of carcinogenesis of PPs have not been established.

With respect to available human data, Roberts et al. refer to clinical studies on clofibrate and gemfibrozil in male subjects as providing evidence of "an absence of a human cancer risk" for fibrate PPs. In a previous review of these data, Ashby et al. (2) noted a small increase in basal cell carcinomas of the skin in gemfibrozil-treated patients, but concluded that the epidemiologic studies on hypolipidemic drugs "are of limited value only, because of the short time periods involved." The World Health Organization trial on the prevention of ischemic heart disease by clofibrate was last updated in 1982 and included 13 years of observation, 5 years during the treatment period plus 8 years of follow-up (3). That study revealed an excess of deaths for nonmalignant diseases of the liver, gall bladder, and intestines in the clofibrate-treated group compared to controls. Because the latency period for clinical manifestation of cancer may be 20 years or more post-exposure, the current data are insufficient to permit a definitive conclusion on the presence or absence of a causal association between exposure to fibrate lipid-lowering drugs and human cancer (4). It is not clear why Roberts et al. claim that "there are no remaining concerns about the human carcinogenic potential of the clinical PPs" inasmuch as a previous review from their laboratory of the same epidemiologic studies found these data to be of limited value due to short study durations (2), and there are no available studies on female cancer risk. Furthermore, in contrast to the view given by Roberts et al., the Physicians' Desk Reference (5) warns

   of the tumorigenicity of clofibrate [and of gemfibrozil (6)] in rodents and
   the possible increased risk of malignancy associated with clofibrate in the
   human.

For DEHP, no epidemiologic studies have been reported.

Roberts et al. also conclude that PPs would "not pose a potential cancer hazard to the human liver." However, as I noted previously (1), it might not be appropriate to expect exact site correspondence for effects of PPs in rodents and humans because of species differences in tissue expression of PPARs. For example, the demonstration of a functional PPAR in human breast cancer cell lines (7) and the finding of enhanced cell proliferation by DEHP in human breast cancer cells (8) indicate a possible breast cancer risk. Furthermore, liver is not the only target organ of tumor induction by hepatic PPs. Several PPs induce tumors of the testis and pancreas in laboratory animals, and tumor induction at these sites occurs without induction of peroxisomes in these affected organs (9).

Roberts et al. claim that available data provide "a plausible mode of carcinogenic action" for PPs, which is based on induction of hepatocyte proliferation and suppression of apoptosis. The latter effects are reported to be absent in cultured human hepatocytes. However, as noted in the Physicians' Desk Reference (5, 6),

   changes in peroxisome morphology and numbers have been observed in humans
   after treatment with several members of the fibrate class, including
   clofibrate, when liver biopsies were compared before and after treatment in
   the same individual.

Several additional issues influence the plausibility of the mode of action for liver carcinogenicity claimed by Roberts et al.:

* Their hypothesis has not been challenged or demonstrated experimentally. For example, no studies have established time- and dose-dependent associations between tumor induction and hepatocyte proliferation and suppression of apoptosis in laboratory animals treated with various PPs.

* It is important to recognize that procedures used to harvest hepatocytes from human livers are usually very different from those used to culture rodent hepatocytes. To evaluate possible functional loss during the time after death until culturing of human hepatocytes, I stressed the critical need to include demonstration of apolipoprotein A-II (ApoA-II) induction in studies on the responsiveness of human hepatocytes to PPs (1). ApoA-II mRNA has been shown to be induced in human hepatocytes via PPAR activation (10). Thus, measurements of ApoA-II induction provide a necessary control to decipher the absence of an effect as being due to the lack of responsiveness to PPs rather than lack of transcriptional function in human cell cultures.

* Because tumor induction by PPs requires long-term exposure, the mechanistic steps in this process most likely involve sustained rather than transient effects. In the case of DEHP and several other PPs, hepatocyte proliferation is not a sustained response with continued exposure (11). Further, cell culture studies are usually performed over a period of a few days and therefore cannot distinguish a sustained response from a transient effect.

* Mechanistic studies in normal human hepatocytes may be focusing on the wrong cell population. Cattley et al. (12) provided data that indicate that the mechanism of liver tumor induction by PPs may be more relevant to changes that are induced in preneoplastic foci rather than in normal hepatocytes.

* Several studies demonstrate that DEHP induces biological effects independent of peroxisome proliferation [e.g., morphologic cell transformation and decreased levels of gap junction intercellular communication (13)]. Roberts et al. ignore the possibility that some of these responses may also contribute to the carcinogenic process. Roberts et al. cite the 11-month study of Wy14,643 in PPAR[alpha] knockout mice (14) as evidence that the carcinogenicity of PPs are mediated by PPAR[alpha]. However, as I noted previously (1), unlike this mouse model, humans do not lack a functional PPAR[alpha], and an 11-month study is not adequate to detect late-developing tumors that might arise by mechanisms independent of PPAR[alpha].

* Roberts et al. cite the finding of an inactive PP response element upstream of the acyl CoA oxidase gene in a sample human population (15) as additional evidence for why humans may be less responsive than rodents to PPs. However, induction of acyl CoA oxidase is not relevant to the suggested carcinogenic mode of action for DEHP and other PPs. Obviously, further work is needed to characterize the expression of all genes that may be affected by PPs in diverse populations.

The inappropriate dismissal of positive animal cancer findings in assessments of human risk could have serious health consequences. Protection of public health requires rigorous testing and validation of mechanistic hypotheses rather than reliance on assertions of plausibility.

REFERENCES AND NOTES

(1.) Melnick RL. Is peroxisome proliferation an obligatory precursor step in the carcinogenicity of di(2-ethylhexyl)phthalate (DEHP)? Environ Health Perspect 109:437-442 (2001).

(2.) Ashby J, Brady A, Elcombe CR, Elliott BM, Ishmael J, 0dum J, Tugwood JD, Kettle S, Purchase IF. Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Hum Exp Toxicol 13:S1-117 (1994).

(3.) WHO cooperative trial on primary prevention of ischaemic heart disease with clofibrate to lower serum cholesterol: final mortality follow-up. Report of the Committee of Principal Investigators. Lancet 2:600-604 (1984).

(4.) Newman TB, Hulley SB, Carcinogenicity of lipid-lowering drugs. JAMA 275:55-60 (1996).

(5.) Atromid-S. In: Physicians' Desk Reference. 55th ed. Montvale, NJ:Medical Economics Data, 2001;3349-3350.

(6.) Lopid. In: Physicians' Desk Reference. 55th ed. Montvale, NJ:Medical Economics Data, 2001;2453-2456.

(7.) Kilgore MW, Tate PL, Rai S, Sengoku E, Price TM. MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response. Mol Cell Endocrinol 129:229-235 (1997).

(8.) Blom A, Ekman E, Johannisson A, Norrgren L, Pesonen M. Effects of xenoestrogenic environmental pollutants on the proliferation of a human breast cancer cell line (MCF-7). Arch Environ Contam Toxicol 34:306-310 (1998).

(9.) Biegel LB, Hurtt ME, Frame SR, O'Connor JO, Cook JC. Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats. Toxicol Sci 60:44-55 (2001).

(10.) Vu-Dac N, Schoonjans K, Kosykh V, Dallongeville J, Fruchart JC, Staels B, Auwerx J. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J Clin Invest 96:741-750 (1995).

(11.) Melnick RL, Huff J. Liver carcinogenesis is not a predicted outcome of chemically induced hepatocyte proliferation. Toxicol Ind Health 9:415-438 (1993).

(12.) Cattley RC, Marsman DS, Popp JA. Age-related susceptibility to the carcinogenic effect of the peroxisome proliferator WY-14,643 in rat liver. Carcinogenesis 12:469-473 (1991).

(13.) Mikalsen SO, Sanner T. Intercellular communication in colonies of Syrian hamster embryo cells and the susceptibility for morphological transformation. Carcinogenesis 14:251-257 (1993).

(14.) Peters JM, Cattley RC, Gonzalez FJ. Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643. Carcinogenesis 18:2029-2233 (1997).

(15.) Woodyatt NJ, Lambe KG, Myers KA, Tugwood JD, Roberts RA. The peroxisome proliferator (PP) response element upstream of the human acyl CoA oxidase gene is inactive among a sample human population: significance for species differences in response to PPs. Carcinogenesis 20:369-372 (1999).

Ronald Melnick
National Institute of Environmental
Health Sciences
Research Triangle Park, NC
E-mail: melnickr@niehs.nih.gov

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