Peroxisomes proliferation, hepatocytes

Chevalier S et al. Proteomic analysis of differential protein expression in primary hepatocytes induced by EG F, tumour necrosis factor alpha or the peroxisome proliferator nafenopin. Eur J Biochem 2000 267 4624-4634. 

Ruyter B., O. Andersen, A. Dehli, A.-K. Ostlrmd Farrants, T. Gjoen, and M.S. Thomassen (1997). Peroxisome proliferator activated receptors in Atlantic salmon Salmo solar)-. Effects on PPAR transcription and acyl-CoA oxidase activity in hepatocytes by peroxisome proliferators and fatty acids. BBA-Lipids and Lipid Metabolism 1348 331-338. 

Three of the compounds evaluated in this volume (di(2-ethylhexyl) phthalate, di(2-ethylhexyl) adipate and cinnamyl anthranilate) are carcinogenic to the liver in mice and/or rats, and have been proposed to act by a mechanism involving peroxisomal proliferation in hepatocytes in those species. The role of peroxisome proliferation in evaluating carcinogenicity in humans has been discussed (lARC, 1995b). When, for any chemical, the relationship between peroxisome proliferation and liver tumours in rats or mice has been established, this should be considered relevant information in the evaluation of the possible risks for cancer in humans, taking into account the following ... 

Peroxisome proliferation may also be demonstrated in vitro in cultured rat and mouse hepatocytes. Many of the known characteristics of peroxisome proliferation in vivo, including increased number and size of peroxisomes, differential induction of peroxisomal enz5mie activities and stimulation of replicative DNA synthesis, have been demonstrated in cultured rat and mouse hepatocytes (lARC, 1995). 

Hepatocytes isolated from male Wistar rats (180-250 g) were treated with 0.2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al., 1982). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. In studies with hepatocytes from male Sprague-Dawley rats (180-220 g), treatment with 0.2 mM mono(2-ethylhexyl) phthalate and 1.0 mM 2-ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold, respectively (Gray et al., 1983). Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and, by ultra-structural examination, to increase numbers of peroxisomes. Hepatocytes were isolated from Wistar-derived rats (180-220 g) and treated for 72 h with 0-0.5 mM mono(2-ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell etal., 1985). Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. In addition, 0-0.5 mM mono(2-ethylhexyl) phthalate and 0-1.0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. In another study with hepatocytes from Wistar-derived rats (180-220 g), metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe Mitchell, 1986). 

Primary hepatocyte cultures may also be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995 Doull et al, 1999). Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g), male Syrian hamsters (70-80 g) and male Dunkin-Hartley guinea-pigs (400-450 g). Treatment with 20-200 0,M mono(2-ethylhexyl) phthalate for 70 h caused strong induction of cyanide-insensitive palmitoyl-CoA oxidation activity in rat hepatocytes (up to 600% of control levels), while no marked effect was observed in Syrian hamster (up to 120% of control) or guinea-pig (down to 80% of control) hepatocytes (Lake et al., 1986). 

Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. Hepatocytes isolated from Wistar-derived rats (180-220 g), male Alderley Park guinea-pigs (400-500 g), male marmosets (350-500 g) and three human liver samples (renal transplant donors) were treated with 0-0.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe Mitchell, 1986). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no induction was observed in guinea-pig or human hepatocytes and only small non-concentration-dependent effects were observed in marmoset hepatocytes. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured... 

Peroxisome proliferators have also been shown to induce replicative DNA synthesis in cultured rodent hepatocytes (lARC, 1995). In contrast, several peroxisome proliferators have failed to induce replicative DNA s mthesis in human hepatocyte cultures (Doull et al., 1999). Hepatocytes were isolated from male Wistar-derived rats and from three human liver samples (liver transplantation donors) and treated with 2-ethylhexanoic acid and some other peroxisome proliferators for 72 h (Elcombe et al, 1996). While 2-ethylhexanoic acid induced replicative DNA s5mthesis in cultured rat hepatocytes, no effect was observed in human hepatocytes. Hepatocytes were isolated from male Fischer 344 rats and three humans and treated in culture with 250-2000 pM mono(2-ethylhexyl) phthalate (Hasmall et al, 1999). Increased peroxisomal (O-oxi-dation (at 250-750 pM), replicative DNA s mthesis (at 500-1000 pM), and inhibition of apoptosis (at 250-1000 pM) were observed in rat hepatocytes. None of these parameters was affected by mono(2-ethylhexyl) phthalate in human hepatocytes. 

Similarly, modulation of hepatocellular proliferation by peroxisome proliferators has been implicated in the mechanism of carcinogenesis. This can theoretically result in increased levels of mutation by increasing the frequency of replicative DNA synthesis as well as increasing the number of hepatocytes at risk. Furthermore, hepatocellular proliferation is probably involved in the promotion of growth of pre-neoplastic hepatocytes. There is clear evidence that di(2-ethylhexyl) phthalate causes acute and sustained hepatocellular proliferation under bioassay conditions which resulted in liver tumours in rats (Marsman et al., 1988). 

Hepatic peroxisome proliferation has not been adequately evaluated in studies of human livers following exposure to di(2-ethylhexyl) phthalate in vivo however, the effect of treatment of human and mouse hepatocytes with di(2-ethylhexyl) phthalate metabolites which are active in rat hepatocytes, as well as other peroxisome proliferators, indicate that humans can reasonably be predicted to be refractory to induction of peroxisome proliferation and hepatocellular proliferation by di(2-ethylhexyl) phthalate. The evidence indicates that the mechanism of peroxisome proliferation induced by di(2-ethylhexyl) phthalate in rat hepatocytes does not operate in humans. 

Hepatic peroxisome proliferation depends on a nuclear receptor, PPARa, to mediate these responses in mice, based on lack of response to peroxisome proliferators in PPARa-deficient mice. In one study with another peroxisome proliferator, WY-14,643, carcinogenesis was shown to be dependent on the same receptor. Oral administration of di(2-ethylhexyl) phthalate failed to elicit markers of peroxisome proliferation in PPARa-deficient mice, while the same treatment elicited this response in normal mice. Metabolites of di(2-ethylhexyl) phthalate caused activation of PPARa-mediated gene expression in mammalian cell co-transfection assays. Differences between responsive rodents and humans in various aspects of PPARa-mediated regulation of gene expression are consistent with the lack of activity of di(2-ethylhexyl) phthalate metabolites in hepatocyte cultures from 12 people studied to date. 

In making its overall evaluation of the carcinogenicity to humans of di(2-ethyl-hexyl) phthalate, the Working Group took into consideration that (a) di(2-ethylhexyl) phthalate produces liver tumours in rats and mice by a non-DNA-reactive mechanism involving peroxisome proliferation h) peroxisome proliferation and hepatocellular proliferation have been demonstrated under the conditions of the carcinogenicity studies of di(2-ethylhexyl) phthalate in rats and mice and (c) peroxisome proliferation has not been documented in human hepatocyte cultures exposed to di(2-ethylhexyl) phthalate nor in the liver of exposed non-human primates. Therefore, the mechanism by which di(2-ethylhexyl) phthalate increases the incidence of hepatocellular tumours in rats and mice is not relevant to humans. 

Gray, T.J.B, Butterworth, K.R., Gaunt, I.F., Grasso, P. Gangolh, S.D. (1977) Short-term toxicity stndy of di-(2-ethylhexyl) phthalate in rats. Food Cosmet. Toxicol., 15, 389-399 Gray, T.J.B, Beamand, J.A., Lake, B.G, Foster, J.R. Gangolli, S.D. (1982) Peroxisome proliferation in cnltmed rat hepatocytes produced by clofibrate and phthalate ester metabolites. 

Heindel, J.J. Powell, C.J. (1992) Phthalate ester effects on rat Sertoli cell function in vitro effects of phthalate side chain and age of animal. Toxicol appl. Pharmacol, 115, 116-123 Hellwig, J., Freudenberger, H. Jackh, R. (1997) Differential prenatal toxicity of branched phthalate esters in rats. Food Cosmel Toxicol, 35, 501-512 Hildebrand, H., Schmidt, U., Kempka, G, Jacob, R., Ahr, H.J., Ebener, C., Goretzki, RE. Bader, A. (1999) An in-vitro model for peroxisome proliferation utilizing primary hepatocytes in sandwich culture. Toxicol. In Vitro, 13, 265-273... 

Kombrast, D.J., Barfknecht, T.R. Ingram, P. (1984) Effect of di(2-ethylhexyl)phthalate on DNA repair and lipid peroxidation in rat hepatocytes and on metabolic cooperation in Chinese hamster V-79 cells. J. Toxicol, environ. Health, 13, 99-116 Kurata, Y., Kidachi, F., Yokoyama, M., Toyota, N., Tsuchitani, M. Katoh, M. (1998) Subchronic toxicity of di(2-ethylhexyl)phthalate in common marmosets lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia. Toxicol. Sci., 42, 49-56... 

Probst, GS. Hill, L.E. (1985) Tests for the induction of DNA-repair synthesis in primary cultures of adult rat hepatocytes. Prog. Mutat. Res., 5, 381-386 Pugh, G, Jr, Isenbeig, J., Kamendulis, L., Clare, L., Brown, W., Lington, A., Ackley, D., Smith, J.H. Klaunig, J. (2000) Absence of liver effects in cynomolgus monkeys treated with peroxisomal proliferators. Toxicol. Sci., 48 (in press)... 

Primary hepatocyte cultures may be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995). The effects of di(2-ethylhexyl) adipate and its metabolites in cultured hepatocytes from rats, mice, guinea-pigs and marmosets have been studied (Cornu et al., 1992). In hepatocytes from each species, the parent compound di(2-ethylhexyl) adipate had no effect on peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity. However, in rat and mouse hepatocytes, the metabolites mono(2-ethylhexyl) adipate, 2-ethylhexanol, 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid at concentrations < 1 mM induced peroxisomal palmitoyl-CoA oxidation. No induction of peroxisomal palmitoyl-CoA oxidation was seen at concentrations < 1 mM for mono(2-ethylhexyl) adipate or < 2 mM for 2-ethylhexanol, 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid in guinea-pig or marmoset hepatocytes (2-ethylhexanol was evaluated only at < 1 mM in marmoset hepatocytes). 

Cultured hepatocytes from non-human primates (marmosets and macaques) and humans have been similarly unresponsive to a variety of peroxisome proliferators (reviewed in Doull et al., 1999). No evaluation of peroxisome proliferation in human hepatocytes treated with di(2-ethylhexyl) adipate metabolites in vitro has been published. The lack of peroxisome proliferation in hepatocytes from marmosets suggests that human hepatocytes also would be unresponsive (Cornu et al., 1992). These negative results were significant in that the same metabolites induced typical induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity in rat and mouse hepatocytes. 

Marked species differences in hepatic peroxisome proliferation have been reported (Ashby et al, 1994 lARC, 1995 Lake, 1995a,b Cattley et al, 1998). No study has yet compared the responsiveness of human versus rodent livers in vivo or hepatocytes in vitro to cinnamyl anthranilate however, a growing body of evidence concerning the molecular basis of peroxisome proliferation indicates that human livers and hepatocytes would be refractory to induction of peroxisome proliferation by cinnamyl anthranilate (Doull et al., 1999). 

A response to exposure to certain foreign compounds, which occurs predominantly in the liver is the phenomenon of peroxisomal proliferation. Peroxisomes (microbodies) are organelles found in many cell types, but especially hepatocytes. Repeated exposure of rodents to certain... 

More recently, however, in cynomologous monkeys, a nonhuman primate, some limited responses to these compounds have been seen with a twofold increase in liver weight, hepatocyte hypertrophy, an increase in peroxisomes (2.7x), and an increase in mitochondria. However, no DNA damage was detected. With humans taking ciprofibrate, only limited peroxisome proliferation was seen, and there was no increase in acyl CoA oxidase. Thus, the question is, "are humans at risk " In order to understand this, the mechanism needs to be understood. 

Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to DEHP. Limited information on hepatic effects in humans exposed to DEHP is available from studies of dialysis patients and cultured human hepatocytes. In one individual there was an increased number of liver peroxisomes after 1 year, but not after 1 month of treatment (Ganning et al. 1984, 1987). A serious limitation of this observation is that repeat biopsies were not obtained from the same patient, so that an appropriately controlled analysis is not possible. Additionally, analysis of liver biopsies from patients receiving other kinds of hypolipidemic drugs has not yielded any evidence for peroxisomal proliferation (Doull et al. 1999). Recognizing some limitations of using primary hepatocytes in vitro because of their tendency to lose some metabolic capabilities (Reid 1990), in cultured human hepatocytes there were no changes in the activities of peroxisomal palmitoyl-CoA oxidase and/or carnitine acetyltransferase when... 

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