Dose-dependent transitions in mechanisms of toxicity: case studies

Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose-response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.     

Molecular analysis of peroxisome proliferation in the hamster

Three novel P450 members of the cytochrome P450 4A family were cloned as partial cDNAs from hamster liver, characterised as novel members of the CYP4A subfamily, and designated CYP4A17, 18, and 19. Hamsters were treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, methylclofenapate (MCP) or Wy-14,643, and shown to develop hepatomegaly and induction of CYP4A17 RNA, and concomitant induction of lauric acid 12- hydroxylase. This treatment also resulted in hypolipidaemia, which was most pronounced in the VLDL fraction, with up to 50% reduction in VLDL-triglycerides; by contrast, blood cholesterol concentration was unaffected by this treatment. These data show that hamster is highly responsive to induction of CYP4A by peroxisome proliferators. To characterise the molecular basis of peroxisome proliferation, the hamster PPARalpha was cloned and shown to encode a 468-amino-acid protein, which is highly similar to rat and mouse PPARalpha proteins. The level of expression of hamster PPARalpha in liver is intermediate between mouse and guinea pig. These results fail to support the hypothesis that the level of PPARalpha in liver is directly responsible for species differences in peroxisome proliferation.     

Phospholipids in X-linked adrenoleukodystrophy white matter: fatty acid abnormalities before the onset of demyelination

Changes in fatty acid composition of complex lipids were analyzed in postmortem white matter from a patient with late onset adrenoleukodystrophy (ALD). The specimen showed three regions with progressive myelin breakdown: morphologically normal white matter; areas with active demyelination and perivascular lymphocyte and macrophage infiltration; and areas with marked gliosis. In the morphologically intact region, cholesterol esters were similar in amount and fatty acid composition to those in control tissue, although marked changes were observed in the actively demyelinating area. Galactolipids in these areas were also similar to those in controls. In contrast, glycerophospholipids were increased in amount and in very long chain fatty acids (VLCFA), which are the hallmark of ALD, at the active edge of the demyelinative lesion and even in the apparently intact sample. Further fractionation of the glycerophospolipids by high performance liquid chromatography showed a significant (up to 39-fold) accumulation of hexacosanoic acid (C26:0) in phosphatidylcholine, but not in other phosphatidyl derivatives. The consistent increases in phosphatidylcholine VLCFA in all samples from the ALD brain, which are postulated to represent progressive stages in the development of the disorder, suggest that phosphatidylcholine may be involved in antigen formation and may underlie an immunological basis for the pathogenesis of ALD.     

Peroxisome proliferation due to di (2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexanoic acid

The dose-response relationships for peroxisome proliferation due to Di (2-ethylhexyl) adipate (DEHA), 2-ethylhexanol (EH), 2-ethylhexanoic acid (EHA) have been investigated in rats and mice. Linear dose-response relationships were observed for induction of cyanide-insensitive palmitoyl CoA oxidation (PCO), used as a enzyme marker of peroxisome proliferation, by DEHA, EH and EHA in both species. Relative liver weights were also increased in a dose related manner. On a molar basis, DEHA was twice as potent as EH or EHA which were equipotent and PCO was stimulated to a greater extent in male mice than in rats or female mice. At doses above 8 mmol/kg/day, EH was toxic to rats (both sexes) and similarly EHA at 13.5 mmol/kg/day lead to the death of female rats. In a attempt to explain the species difference in carcinogenicity of DEHA previously reported, we also used Fischer 344 rats and B6C3F1 mice. DEHA administration (2.5 g/kg/day) to Fischer 344 rats and B6C3F1 mice lead to toxicity in female rats. Relative liver weights were increased in a dose related fashion by DEHA administration to both rats and mice, PCO but not catalase was markedly increased (up to 15 fold in male rats). Light microscopy examination indicated some glycogen loss, a dose related hypertrophy and increased eosinophilia in both rats and mice. Electron microscopy confirmed peroxisome proliferation accompanied by a marked reduction of lipid in the centrilobular hepatocytes. These data suggest EHA to be the proximate peroxisome proliferator derived from DEHA. These data indicate a higher sensitivity for Fischer 344 rats than B6C3F1 mice to hepatic peroxisome proliferation due to DEHA and ratio of PCO activity and catalase activity data suggest that more hydrogen peroxide (H₂O₂) could escape from peroxisomes in male Fischer 344 rats than B6C3F1 mice. These data obtained with B6C3F1 mice and Fischer 344 rats are not agreement with the carcinogenicity bioassays previously reported showing an incidence of hepatic tumours only in B6C3F1 mice.     

Reciprocal expression of peroxisome proliferator-activated receptor-gamma and cyclooxygenase-2 in human term parturition

OBJECTIVE: The objective of this study was to test the hypothesis that peroxisome proliferator activated receptor-gamma (PPAR-gamma) is expressed in intrauterine tissues before active term human parturition, and that its repression is associated with up-regulation of cyclooxygenase-2 (COX-2). STUDY DESIGN: Specimens were collected from women with term singleton pregnancies after spontaneous labor or cesarean section before labor, prepared for immunoblot and immunohistochemical analysis, and probed for PPAR-gamma or COX-2. RESULTS: PPAR-gamma expression was prominent in fetal membranes and placenta before active labor. After labor, PPAR-gamma expression was significantly reduced in fetal membranes, but not in placenta. The ratio of COX-2:PPAR-gamma was significantly elevated in fetal membranes with labor. PPAR-gamma immunostaining was prominent in syncytiotrophoblast, extravillous cytotrophoblasts, and cells of the amnion and chorion. COX-2 immunostaining was abundant in the amnion and rare in the placenta. CONCLUSION: PPAR-gamma is highly expressed in term intrauterine tissues. In fetal membranes, PPAR-gamma levels are reduced once active labor commences, coincidental with a relative increase in COX-2 expression.     

α-Lipoic acid-based PPARγ agonists for treating inflammatory skin diseases

Novel thiazolidinedione derivatives of the potent antioxidant, -lipoic (thioctic, 1,2-dithiolane) acid, were prepared. The prototype N-(2-{4-[2,4-dioxo(1,3-thiazolidin-5-yl)methyl]phenoxy}ethyl)-5-(1,2-dithiolan-3-yl)-N-methylpentanamide (designated BP-1003), and dithioester derivatives thereof were shown to be potent activators of peroxisome proliferator-activated receptor gamma (PPAR) (EC₅₀ range 15–101 nM) and modest activators of PPAR (EC₅₀ 5 M). Both the relatively hydrophobic dithiolane prototype, BP-1003, and its water-soluble dithioglycinate derivative, BP-1017, were shown to inhibit the proliferation of human keratinocytes and suppress the production of interleukin-2 by human peripheral lymphocytes to a greater extent than the antidiabetic thiazolidinedione, rosiglitazone. Both oral and topical administration of BP-1017 showed significant antiinflammatory effects in the oxazolone-sensitized mouse model of allergic contact dermatitis (ACD). These findings suggest that water-soluble lipoic acid-based thiazolidinediones may be efficacious as oral and topical agents for treating inflammatory skin conditions such as contact dermatitis, atopic dermatitis, and psoriasis.Abbreviations ACD Allergic contact dermatitis - ATCC American Type Culture Collection - FRET Fluorescence resonance transactivation - LBD Ligand binding domain - LDH Lactate dehydrogenase - PBMC Peripheral blood mononuclear cells - PHA Phytohemagglutinin - PPAR Peroxisome proliferator-activated receptor-gamma - TZD Thiazolidinedione     

Sister chromatid exchange induction by the herbicide 2,4-dichlorophenoxyacetic acid in chick embryos

As genetic damage may result from exposure to agricultural chemicals, it seemed appropriate to assess the genotoxic potential of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used broad-leaf herbicide, using a test system that may provide some indications on the genetic risk to animal species in the wild. In the present study, sister chromatid exchange (SCE) induction and cell cycle kinetics alterations by 2,4-D in 4-day old chick embryos were evaluated. Both a commercial herbicide formulation containing 37% 2,4-D isooctyl ester as active ingredient and pure 2,4-D were tested. Chick embryos were treated with 0, 0.5, 1, 2, or 4 mg 2,4-D. Test solutions were applied to the inner shell membrane on day 0 of incubation. Either commercial formulation or pure 2,4-D induced a dose-related increase in SCE frequency over the concentration range from 0 to 4 mg/embryo. Significantly higher SCE frequency was seen for the 4-mg group of embryos treated with the commercial product. A slightly higher SCE value was observed for the vehicle group (acetone-treated embryos) compared with the negative controls (untreated embryos). Significant inhibition of cell cycle progression was evident in both experimental groups and was generally dose related. The extent of changes in cell kinetics was similar in both groups, although somewhat more marked in the group treated with pure 2,4-D. The present findings corroborate the positive results from recent in vivo rodent studies.     

Role of cytokines in non-genotoxic hepatocarcinogenesis: cause or effect?

Chemicals with the potential to cause cancer through damaging DNA can be readily identified in a range of in vitro screens that detect genotoxicity. However, many carcinogens are non-genotoxic yet cause rodent tumours, particularly in the liver. Some non-genotoxic carcinogens such as the peroxisome proliferators (PPs) act directly to cause liver growth and proliferation, whereas others such as carbon tetrachloride cause liver damage, followed by regenerative hyperplasia. Current data support a role for cytokines such as tumour necrosis factor alpha (TNFalpha) and interleukin 1 (IL1) in hepatocarcinogenesis. However, these data give rise to conflicting hypotheses; in some experimental models, TNFalpha appears to mediate damage, whereas in others it is postulated to play a role in tissue repair. Recently, we have shown that TNFalpha acting via TNFalpha receptor 1 and p38 MAP kinase suppresses hepatocyte apoptosis. However, when new protein synthesis is disabled, TNFalpha becomes a death signal. An understanding of the role of cytokines in rodent hepatocarcinogenesis will allow the development of markers that can be used to identify, at an early stage, those chemicals with the potential to induce rodent tumours.