Hillary M. Carpenter

Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout

Background Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. Objectives In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. Methods We fed aflatoxin B1 or sham-initiated animals 200–1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17β-estradiol (E2, a known tumor promoter) in the diet for 14 days. Results PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal β-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E2 by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E2. Conclusions These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation.

Hexachlorobenzene-induced porphyria in Japanese quail: effect of pretreatment with phenobarbital or β-naphthoflavone

In an effort to determine the role that metabolism by the cytochrome P-450 system plays in the development of hexachlorobenzene (HCB)-induced porphyria, Japanese quail were pretreated with either beta-naphthoflavone (BNF) or phenobarbital (PB) and then treated with HCB. PB or BNF pretreatment appeared to have no effect on the response of quail hepatic enzymes to HCB. There were no differences between the two groups in either the content of cytochrome P-450 or the activities of NADPH-cytochrome c reductase, glutathione transferase (microsomal or cytosolic), ethoxycoumarin-O-deethylase or ethoxyresorufin-O-deethylase following HCB treatment. These pretreatments did, however, markedly influence the development of porphyria in quail. BNF-treated birds had higher delta-aminolevulinic acid-synthetase (ALA-S) activities and developed porphyria much more rapidly than birds treated with HCB alone. Birds pretreated with PB did not exhibit porphyria even following 10 days of HCB. Although the ALA-S activities in this group were elevated slightly following HCB, they were about one-half of those seen in the BNF-pretreated HCB-treated group. These results may reflect a difference between the PB and BNF groups in the production of a porphyrogenic metabolite of HCB.

Effect of ethoxyquin on the toxicity of the pyrrolizidine alkaloid monocrotaline and on hepatic drug metabolism in mice.

Abstract Diets containing the antioxidant ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) were fed to female CD-1 mice for 10–38 days to assess their effects on monocrotaline toxicity, liver glutathione levels and hepatic drug metabolizing enzyme activities. Dietary ethoxyquin (0.25%) protected the mice against lethality as well as acute hepatotoxicity of monocrotaline as measured by the levels of alanine aminotransferase and asparate aminotransferase in plasma. Other feed additives with antioxidant properties such as vitamin C, vitamin E or selenium had no protective effect against monocrotaline lethality and hepatotoxicity. Dietary cysteine (1%) also protected mice against the lethality but not the acute hepatotoxicity of the alkaloid. With the exception of ethoxyquin, none of the other feed additives increased liver glutathione levels (mg/liver). Glutathione S-transferase activity was significantly increased by either dietary ethoxyquin or cysteine using chlorodinitrobenzene as substrate. Dietary ethoxyquin produced an increase in hepatic cytochrome P-450 content and an increase in the in vitro conversion of monocrotaline to pyrrole metabolites by liver microsomes. However, there was no effect of the feed additive on the activity of aminopyrine demethylase and on the concentration of pyrroles found in the liver 2 or 24 h after monocrotaline administration. Since ethoxyquin protected mice against monocrotaline lethality and hepatotoxicity despite no reduction in the in vivo activation of monocrotaline, the mechanisms involved are most probably a result of increased detoxication processes partly because of increased liver glutathione levels.

The effect of thermal acclimation on the activity of arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss)

1. The possibility that temperature acclimation (to 10 or 18 degrees C for 28 days) would alter the cytochromes P-450 of rainbow trout was addressed. 2. The specific content of LM4b (P-450 IA1), the trout isozyme responsible for activation of polynuclear aromatic hydrocarbons, was lower in 18 degrees C fish than it was in 10 degrees C fish. 3. Kinetic analysis of aryl hydrocarbon hydroxylase indicated that, while thermal acclimation caused no change in Vmax, it lowered the apparent Km of this enzyme for benzo[a]pyrene when assayed at acutely shifted temperatures. 4. Thermal acclimation of fish may have significance when feral populations are subjected to acute temperature shifts.

Modulation of 7,12-dimethylbenz[a]anthracene disposition and hepatocarcinogenesis by dieldrin and chlordecone in rainbow trout.

The present study examined whether modified xenobiotic transport, resulting from chlordecone (CD) or dieldrin pretreatment, would alter polycyclic aromatic hydrocarbon (PAH) or organochlorine (OC) target organ doses and subsequent tumor organospecificity or incidence rates in rainbow trout. Additionally, the potential for exposure to dieldrin or CD, following PAH exposure, to enhance tumor incidence was assessed. Evaluation of CD pretreatment effects on [14C]CD disposition in trout was conducted following two i.p. (0-15 mg/kg) and two dietary (0-0.4 mg/kg/d) pretreatment regimes. To assess the influence of OC pretreatment on cancer induced by the PAH 7,12-dimethylbenz[a]anthracene (DMBA), juvenile trout were fed control, CD (0.1, 0.4 mg/kg/d), or dieldrin (0.1, 0.3 mg/kg/d) diets for 9 wk, received a waterborne [3H]DMBA exposure (1 mg/L, 20 h), and resumed control, CD, or dieldrin diets for 33 wk. [3H]DMBA disposition and hepatic [3H]DMBA binding were examined immediately and 24 h after exposure. Hepatic and stomach tumor incidences were determined 33 wk after DMBA exposure. CD pretreatment did not influence [14C]CD or [3H]DMBA hepatic concentrations, hepatic [3H]DMBA DNA binding, or hepatic/stomach tumor incidence. It did, however, elevate bile [14C]CD and [3H]DMBA concentrations. Postinitiation exposure to CD weakly enhanced DMBA-induced hepatic tumor incidence at the low but not the high CD dose. Dieldrin pretreatment did not influence stomach [3H]DMBA equivalents or stomach tumor incidence but did cause an elevation in biliary and hepatic concentrations of [3H]DMBA equivalents. [3H]DMBA binding to liver DNA was significantly increased and hepatic tumor incidence was elevated by dieldrin pretreatment. Dieldrin treatment following DMBA initiation did not enhance hepatic or stomach tumor incidence. Ecoepidemiology studies, to date, have reported correlations between the co-occurrence of PAHs and OCs and elevated tumor incidence in feral fish, but cause-and-effect relationships have been difficult to establish. The results of the present study confirm that OCs, such as dieldrin and CD, play a role in modifying PAH-induced carcinogenesis in fish.

Alterations in lipid peroxidation, antioxidant enzymes, and carcinogen metabolism in liver microsomes of vitamin E-deficient trout and rat

Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (18-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in vivo were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin E-deficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [3H]-(+)-benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants.

Hexachlorobenzene‐induced porphyria in Japanese quail: Changes in microsomal enzymes

Hexachlorobenzene (HCB) was administered orally (500 mg/kg d) for 1, 2, 5, or 10d) to sexually mature Japanese quail to compare altered hepatic porphyrin levels with changes that occur in hepatic xenobiotic metabolizing enzymes. Porphyrin levels rapidly increased following the administration of HCB (three times control levels after a single dose of HCB), and birds began to develop porphyria (i.e., porphyrin levels were at least 10 times higher than controls) following 5 d of treatment. Following 10 d of HCB treatment, 3 of 4 treated quail were porphyric. Coincident with the HCB-induced disruption of the heme biosynthetic pathway were increases in various hepatic constituents. Changes included elevation of microsomal protein concentrations and increases in the specific content of cytochrome P-450, in the activities of aryl hydrocarbon hydroxylase (AHH), biphenyl hydroxylase (BPH), ethoxyresorufin-O-deethylase (EROD), and ethoxycoumarin-O-deethylase (ECOD), and in cytosolic and microsomal glutathione S-transferase (GSH-t) levels. In addition, the lambda max of the CO versus CO-reduced absorption spectra of hepatic microsomes from HCB-dosed birds showed a hypsochromic shift of 450 to 448 nm. The activity of NADPH-cytochrome P-450 reductase was increased following 10 d of HCB, and the activity of epoxide hydrolase was increased following 5 d of HCB. Most of these changes occurred with a single HCB treatment, and no further alterations developed in the nature of the response with repetitive dosing. Only weight loss, increased cytochrome P-450 content, and increases in GSH-t activity occurred simultaneously with the induction of porphyria.

Ultrastructural, protein, and lipid changes in liver associated with chlordecone treatment of mice

Pretreatment of mice with chlordecone (CD) reduced hepatic accumulation of a subsequent dose of [14C]CD without significantly changing [14C]CD biotransformation. To determine if CD-induced changes in hepatic [14C]CD accumulation were coincident with altered cell composition, we examined the effects of CD on hepatic protein and lipid content, on fatty acid profiles of liver and kidney, and on the ultrastructure of hepatocytes. SDS-polyacrylamide gel electrophoresis detected an apparent CD dose-related increase in a microsomal protein with a molecular weight of about 23 kDa. Total liver or kidney lipid contents were not altered by CD but relative amounts of several hepatic fatty acids were changed. CD caused marked hepatic mitochondrial swelling, increased amounts of endoplasmic reticulum, apparently increased numbers of peroxisome-like structures, and decreased numbers of lipid droplets in cytoplasm of hepatocytes. Numbers of lipid droplets were not decreased in perisinusoidal fat storage cells. In addition, the numbers of cytoplasmic lipoprotein vesicles were apparently increased in some hepatocytes. Overall these changes indicated an increased hepatocyte secretory activity and suggested that CD changed hepatocellular lipid transport, storage, and metabolism pathways.

Chlordecone Pretreatment Alters [14C]Chlordecone and [14C]Cholesterol Transport Kinetics in the Perfused Rat Liver,

Previous work demonstrated that pretreatment of mice with low doses of the organochlorine insecticide chlordecone (CD) altered the tissue disposition of a subsequent [14C]CD or [14C]cholesterol challenge dose. The profile of these changes was consistent with the induction of a protein integral to hepatic CD/cholesterol turnover. The present study was undertaken to confirm similar in vivo effects in the rat and to analyze potential CD-induced changes in hepatic transport kinetics in the perfused rat liver. For in vivo experiments, male, Sprague-Dawley rats were treated with CD (5, 15, or 40 mg/kg) and challenged 3 or 7 days later with a 5 mg/kg [14C]CD tracer dose. Rats challenged 3 days after treatment and evaluated 16 hr later showed a dose-dependent decrease in hepatic [14C]CD relative to controls. This decrease could not be attributed to alterations in liver mass or total liver lipid. For kinetics studies, rats received 15 mg/kg CD and livers were perfused 3 days later. Following a brief (5-7 min) single-pass perfusion, the perfusate was replaced with recirculating buffer containing albumin-bound [3H]oleic acid or high-density lipoprotein-bound [14C]CD or [14C]cholesterol. Livers from pretreated animals had significantly decreased rates of [14C]CD and [14C]cholesterol uptake. Efflux of [14C]CD and biliary excretion of [14C]cholesterol were increased. No changes were observed in uptake or biliary excretion of [3H]oleic acid. SDS-PAGE of hepatic cytosol revealed an enhanced band intensity corresponding to a M(r) of 25,600 in livers from pretreated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the porphyrinogenic action of 1,2,4-trichlorobenzene in birds.

The porphyrinogenic action of 1,2,4-trichlorobenzene (TCB) was examined in 17-day-old embryos, day-old chicks, 18-day-old chickens and adult Japanese quail. The quail was found to be the most sensitive species towards TCB induced porphyria whereas the chick embryo was totally non-responsive. The liver porphyrins of Japanese quail were increased in a dose-dependent manner 1 day after TCB. Elevation in porphyrin levels in quail was associated with comparable increases in delta-aminolevulinic acid synthetase (ALA-S) activity 1 day after TCB treatment. In contrast, ferrochelatase activity was found to be unchanged 1 day after TCB. Multiple administration of TCB produced only a slight increase in liver porphyrin levels and ALA-S activity in quail. However, there was a marked induction in ferrochelatase activity suggesting increased porphyrin turnover. Liver glutathione and glutathione S-transferase activity were also significantly increased following repeated administration of TCB in quail, which could indicate an enhancement of detoxication of reactive metabolites of TCB. Thus, it is suggested that the inability of low multiple doses of TCB to cause porphyria in Japanese quail may be related to the low responsiveness of ALA-S but high inducibility of ferrochelatase liver GSH and glutathione S-transferase.