Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non‐genotoxic hepatocarcinogens with cytochrome P450‐inducible potency

Abstract

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP-inducible potency. In the present study, we investigated 8-hydroxydeoxyguanosine (8-OHdG) levels, in vivo mutagenicity and glutathione S-transferase placental form (GST-P)-positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time-dependent and statistically significant increases in 8-OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)-positive hepatocytes at 4 weeks, while no increase in 8-OHdG levels was found in PhB-treated rats. No changes in mutant frequencies of gpt and red/gam (Spi -) genes in liver DNA from PBO-or PhB-treated rats were observed at 4 or 13 weeks. A 13-week exposure to either PBO or PhB did not affect the number and area of GST-P-positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8-OHdG in PBO-treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8-OHdG-related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation. (Cancer Sci 2010; 101: 2525-2530 O xidative stress induces cell proliferation due to either activation of various transcriptional genes related to the cell cycle or to site-specific toxicity. As such, oxidative stress may take part in non-genotoxic carcinogenesis.(1,2) Exposure to exogenous chemicals are able to yield oxidative stress in the body by various ways such as their oxidizing properties, (3) production of radicals through a redox cycle in their metabolism, (4) depletion of glutathione resulting from utilization for their conjugation, (5) and their cytochrome P450 (CYP)-mediated metabolizing pathways.(6) All of these mechanisms for generating oxidative stress are characterized by having site specificity. In addition, all of the above cases except oxidizing properties require in vivo metabolism to produce oxidative stress, which may be the major reason why such chemicals fail to show genotoxicity in conventional mutagenicity tests. The CYP act on a wide variety of chemicals, and CYP involvement in the production of reactive oxygen species during substrate oxidation is well documented.(7-9) However, since the amount of free radicals generated through the normal catalytic cycle is small, (10) induction of CYP might be necessary for any associated oxidative properties to play a role in carcinogenesis in vivo. Various chemicals with CYP-inducible properties demonstrate hepatocar...

Dammar resin, a non-mutagen, inducts oxidative stress and metabolic enzymes in the liver of gpt delta transgenic mouse which is different from a mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline

“…However, while the Dammar resin fed group had increased 8-OHdG levels, there was no evidence suggestive of direct DNA damage, i.e., the Dammar resin fed group did not have increased DNA mutations. Therefore, our results suggest that the production of reactive oxygen species resulting from increased expression and activity of P450 enzymes, while causing chemical damage to DNA bases, is insufficient to cause permanent gene mutations; this result is consistent with a previous study [15].…”

“…Thus, we hypothesize that intranuclear oxidative stress could play a role in Dammar resin hepatocarcinogenicity by altering gene expression as a result of 8-OHdG adduct formation, but not by inducing DNA mutation. Therefore, consistent with a previous study by Tasaki et al [15], it is reasonable to suggest that the hepatocarcinogenicity of Dammar resin observed in our previous long-term experiment with rats (unpublished data) may not be caused by DNA mutations, but rather by non-genotoxic mechanisms: Promotion of cell proliferation, induction of P450s, detoxification and antioxidant enzymes, increased intranuclear oxidative stress and other gene products. Although the mechanisms responsible for the carcinogenicity of Dammar resin remain to be further defined, our findings support the suggestion that its carcinogenicity does not necessarily correlate with in vivo mutagenicity [1].…”

“…Various chemicals with P450-inducible properties demonstrate hepatocarcinogenicity without overt genotoxicity [12][13][14], which suggests that reactive oxygen species resulting from increased activity of P450s enzymes may play a role in the carcinogenic activity of these chemicals. Researchers have hypothesized that non-genotoxic hepatocarcinogens with P450-inducible capability may cause oxidative damage to liver DNA, which might subsequently take part in inducing carcinogenesis [15].…”

Dose-dependent difference of nuclear receptors involved in murine liver hypertrophy by piperonyl butoxide

“…In the induction of liver hypertrophy, PBO increases expression of liver metabolizing enzymes such as CYP1A1, 1A2, 2B1/2, 3A and 4A in rats Watanabe et al, 1998;Tasaki et al, 2010;Morita et al, 2013) and CYP1A1, 1A2 and 2B10 in mice. Among them, CYP1A1 induction in mice was reported to be mediated by the aryl hydrocarbon receptor activation (Adams et al, 1993;Ryu et al, 1996;Muguruma et al, 2006).…”

Possible Carcinogenic Mechanisms Underlying Renal Carcinogens in Food

“…However, positive outcomes in mutagenicity and carcinogenicity tests may not always imply the involvement of genotoxic mechanisms in carcinogenesis. Recently, transgenic rodents carrying reporter genes such as lacI, lacZ, and gpt have been developed 2,3) . Reporter gene mutation assays that consider the absorption, distribution, metabolism and excretion of test chemicals may provide information regarding in ©2014 Food Safety Commission, Cabinet Office, Government of Japan doi: 10.14252/foodsafetyfscj.2014015 vivo mutagenicity at carcinogenic target sites.…”