Human relevance of rodent liver tumors: Key insights from a Toxicology Forum workshop on nongenotoxic modes of action.

The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.

Intestinal bioavailability and biotransformation of 3,3',4,4'-tetrachlorobiphenyl (CB 77) in in situ preparations of channel catfish following dietary induction of CYP1A.

Previous studies with the catfish in situ perfused intestinal preparation have demonstrated a significant decline in the intestinal bioavailability of a coplanar polychlorinated biphenyl (PCB), 3,3',4,4'-tetrachlorobiphenyl (CB 77)(14C-TCB) dose in animals pre-exposed in vivo to TCB. This response was accompanied by CYP1A induction in the intestine, but little effect upon the oxidative metabolism of the subsequent in situ dose of [14C]-TCB. To ascertain the basis of these responses and the intestine specific contributions, the intestinal bioavailability and metabolism of [14C]-TCB were examined in the in situ intestinal preparation following in vivo exposure to beta-naphthoflavone (BNF; 0, 10 or 50 mg BNF/kg diet for 10 days), BNF was selected as a known inducer of CYP1A and as a compound with a structure unlikely to influence or directly partake in diffusion based TCB concentration gradients. Appreciable amounts of [14C]-TCB molar equivalents (Meq) reached the perfused circulation of the intestinal preparation for all treatments. While BNF pre-exposure elicited induction of CYP1A activities aryl hydrocarbon hydroxylase (AHH) (9.2-12.5-fold) and elicited modest morphological changes (muciparous) in the intestine these changes were not associated with alterations in [14C]-TCB Meq bioavailability. [14C]-TCB metabolism in the intestinal mucosa ranged between 0.54 and 1.27%, for all treatments. As with bioavailability, intestinal metabolism of [14C]-TCB was not significantly influenced in either extent or profile by induction of CYP1A activity as associated with BNF treatment. Four metabolites were found in mucosal sample extracts of which three were tentatively identified as 2-OH-TCB, 4-OH-3,3',4',5-TCB, and 4,4'-diOH-3,3',5,5' tetrachlorobiphenyl. A fourth unknown metabolite presented chromatographic characteristics suggestive of another dihydroxylated metabolite. These data when examined alone and compared to the literature suggest that the intestine may metabolize [14C]-TCB slowly and independent of CYP1A, resulting in somewhat different profiles than published for other organs. In addition, it is likely that previous [14C]-TCB bioavailability findings in the perfused intestine may be based on TCB concentration gradients rather than biotransformation.

Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal.

We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthys flesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6 x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice.

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.

alpha 2u-globulin nephropathy and renal tumors in national toxicology program studies.

Chemically induced renal neoplasms in male rats, developed coincident with alpha(2u)-globulin nephropathy, are not considered predictive of risk to humans by the International Agency for Research on Cancer (IARC) and the U.S. Environmental Protection Agency. Criteria have been defined to establish the role of alpha(2u)-globulin nephropathy in renal carcinogenesis, based on a proposed mode of action involving sustained tubular cell proliferation resulting from alpha(2u)-induced nephropathy, with consequent development of neoplastic lesions. Recent NTP studies demonstrated inconsistencies with this proposed mechanism, including in some cases, far weaker kidney tumor responses than expected based on the extent of alpha(2u)-globulin nephropathy. NTP studies with decalin, propylene glycol mono-t-butyl ether and Stoddard solvent IIC included extended evaluations of alpha(2u)-related nephropathy, and were thus used in assessing the linkage between key events in 90-day studies with renal tumors in 2-year studies. This review revealed no or at best weak associations of tumor responses with renal alpha(2u)-globulin concentrations, indices of cell turnover, or microscopic evidence of alpha(2u)-associated nephropathy in prechronic studies. While tumor responses corresponded somewhat with a measure of cumulative alpha(2u)-associated nephropathy (linear mineralization of the papilla) at the end of the 2-year studies, the severity of chronic nephropathy was generally in best agreement with the pattern of tumor response. These results suggest that while alpha(2u)-globulin nephropathy may contribute to the renal tumor response, the critical component(s) of the nephropathy most closely associated with the development of tumors could not be clearly identified in this review.

Influence of functional group substitutions on the carcinogenicity of anthraquinone in rats and mice: analysis of long-term bioassays by the National Cancer Institute and the National Toxicology Program.

The carcinogenic activities of anthraquinone and six derivatives were compared and contrasted. Studies included representatives of amino, alkyl, nitro, hydroxy, or halogen-containing anthraquinones, with the purpose of uncovering general structure-activity relationships. Anthraquinone, 2-aminoanthraquinone, 1-amino-2-methylanthraquinone, 2-methyl-1-nitroanthraquinone,1-amino-2,4-dibromoanthraquinone, 1,4,5,8-tetraaminoanthraquinone, and 1,3,8-trihydroxy-6-methylanthraquinone (of varying purities) were administered via feed to Fischer 344/N rats and B6C3F, mice. In rats, anthraquinone induced tumors in the liver, kidney, and urinary bladder. A 2-amino substitution narrowed the carcinogenicity to the liver, while multiple amino substitutions led to a carcinogenic response in the urinary bladder alone. A methyl substitution ortho to a 1-aminogroup preserved the hepatic and renal neoplasms seen with the parent anthraquinone, but did not induce urinary bladder tumors; amino or bromo substitutions para to a 1-amino group were related to urinary bladder neoplasms. The intestine may have been a target organ for bromine-substituted anthraquinones. The presence of a nitro group altered the targets of carcinogenicity, and skin tumors may have been associated with this particular functional group in both rats and mice. Over-all for mice, the findings were somewhat different and limited by the small number of common target organs. The parent anthraquinone was clearly carcinogenic only to the liver. There were no other effects of single amino substitutions, in the presence or absence of an additional methyl group, on the carcinogenicity or the site of carcinogenesis of anthraquinone in mice. Multiple amino substitutions diminished, while bromine substitutions enhanced the carcinogenicity induced by anthraquinone and extended the target organs to include forestomach and lung.

Topical application of representative multifunctional acrylates produced proliferative and inflammatory lesions in F344/N rats and B6C3F1 mice, and squamous cell neoplasms in Tg.AC mice.

Widespread human exposure to multifunctional acrylates is of concern, due to their inherent reactivity and irritating properties. Trimethylolpropane triacrylate (TMPTA) and pentaerythritol triacrylate (PETA) are industrially important representatives of multifunctional acrylates. The current studies characterized the toxicity of 3-month topical administration of technical grade TMPTA and PETA in F344/N rats and B6C3F1 mice, and evaluated the carcinogenic potential of TMPTA and PETA in hemizygous Tg.AC (v-Ha-ras) transgenic mice. Administration of 0.75, 1.5, 3, 6, and 12 mg/kg TMPTA and PETA for 3 months resulted in hyperplastic, degenerative, and necrotic lesions, accompanied by chronic inflammation of the skin, with severities generally increasing with dose. Lesions were slightly more severe in rats, when compared with mice, and illustrate the irritant potential of TMPTA and PETA. A similar dosage regimen was used for the 6-month study with Tg.AC mice. Topical application of TMPTA and PETA to Tg.AC mice showed dose-dependent increases in squamous cell papillomas at the site of application, with decreases in the latency of their appearance in mice receiving 3 mg/kg or greater. Papillomas, the reporter phenotype in Tg.AC mice, were accompanied by a few squamous cell carcinomas, along with hyperplastic and inflammatory lesions. Although chronic inflammation might have contributed to the development of the skin lesions, the dose-related nature of the induction of the skin papillomas in Tg.AC mice by TMPTA and PETA may reflect a potential for carcinogenicity.

Variability in aflatoxin B(1)-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver.

Studies of transplacental transfer of aflatoxin B(1) (AFB(1)) suggest that the developing human fetus may be a sensitive target for AFB(1) injury. Because AFB(1) requires metabolic activation to the reactive AFB(1)-8,9-exo-epoxide (AFBO) to exert its carcinogenic effects, ontogenic and interindividual differences in AFB(1) biotransformation enzymes may underlie susceptibility to AFB(1)-induced cell injury. The present study was initiated to compare the rates of in vitro AFB(1)-DNA and AFB(1)-protein adduct formation among a panel of 10 adult and 10 second-trimester prenatal livers and to examine the relationship among AFB(1) metabolizing enzyme expression and AFB(1) binding. Mixtures of cytosolic and microsomal proteins from prenatal and adult livers catalyzed the formation of AFB(1)-DNA and AFB(1)-protein adducts at relatively similar rates, although greater individual variability in AFB(1) adduct formation was observed in adult tissues. Extensive interindividual variation among adult tissues was observed in the expression of the AFB(1) activation enzymes cytochrome P4501A2 (CYP1A2), CYP3A4/5, and lipoxygenase (LO). Prenatal CYP3A7 expression was also highly variable. LO expression was eightfold higher in prenatal liver tissues than adults, whereas the expression of the AFBO detoxification enzyme microsomal epoxide hydrolase was twofold higher in adult liver. The levels of the polymorphic glutathione S-transferase M1 (hGSTM1-1), which may potentially protect against AFBO injury, were higher in the hGSTM1-1-expressing tissues of adults in relation to prenatal livers. In general, there was not a strong relationship among AFB(1)-DNA or AFB(1)-protein adduct formation and expression levels of individual AFB(1) metabolizing enzymes. In summary, despite the presence of marked individual and ontogenic differences in the expression of AFB(1) metabolizing enzymes, human second trimester prenatal liver tissues compared to adults do not exhibit a marked sensitivity to the in vitro formation of macromolecular AFB(1) adducts.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells.

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic alpha,beta-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST-4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 microM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.