Expressions of hepatic genes, especially IGF-binding protein-1, correlating with serum corticosterone in microarray analysis.

Microarray technology was evaluated for usefulness in assessing relationships between serum corticosterone and hepatic gene expression. Nine pairs of female Swiss mice were chosen to provide a wide range of serum corticosterone ratios; cDNA microarray analysis (approximately 8000 genes) was performed on their livers. A statistical method based on calculation of 99% confidence intervals discovered 32 genes which varied significantly among the livers. Five of these ratios correlated significantly with serum corticosterone ratio, including tyrosine aminotransferase, stress-induced protein, pleiotropic regulator 1 and insulin-like growth factor-binding protein-1; the latter has a potential role in cancer development. Secondly, linear regression of gene expression vs corticosterone ratios was screened for those with r> or =0.8 (P<0.01), yielding 141 genes, including some known to be corticosterone regulated and others of interest as possible glucocorticoid targets. Half of these significant correlations involved data sets where no microarray ratio exceeded +/- 1.5. These results showed that microarray may be used to survey tissues for changes in gene expression related to serum hormones, and that even small changes in expression can be of statistical significance in a study with adequate numbers of replicate samples.

Differential zinc and DNA binding by partial peptides of human protamine HP2.

The Zn(II) binding by partial peptides of human protamine HP2: HP2(1-15); HP2(1-25), HP2(26-40), HP2(37-47), and HP2(43-57) was studied by circular dichroism (CD). Precipitation of a 20-mer DNA by these partial peptides and the effects of Zn(II) thereon were investigated using polyacrylamide gel electrophoresis (GE). The results of this study suggest that reduced HP2 (thiol groups intact) can bind Zn(II) at various parts of the molecule. In the absence of DNA, the primary Zn(II) binding site in reduced HP2 is located in the 37-47 sequence (involving Cys-37, His-39, His-43, and Cys-47), while in the presence of DNA, the strongest Zn(II) binding is provided by sequences 12-22 (by His-12, Cys-13, His-19, and His-22) and 43-57 (His-43, Cys-47, Cys-53, and His-57). In its oxidized form, HP2 can bind zinc through His residues of the 7-22 sequence. Zn(II) markedly enhances DNA binding by all partial peptides. These findings suggest that Zn(II) ions may be a regulatory factor for sperm chromatin condensation processes.

Stray Cu(II) may cause oxidative damage when coordinated to the -TESHHK- sequence derived from the C-terminal tail of histone H2A.

CH(3)CO-Thr-Glu-Ser-His-His-Lys-NH(2), a hexapeptide representing the 120-125 sequence of histone H2A, coordinates Cu(II) ions efficiently. Monomeric complexes are formed. In the major complex at physiological pH, CuH(-1)L, Cu(II) is coordinated equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, and axially through the imidazole nitrogen of the His-5 residue. This complex reacts with H(2)O(2) and the resulting reactive oxygen intermediate efficiently oxidizes 2'-deoxyguanosine. The underlying mechanism involves the formation of Cu(III) and a metal-bound hydroxyl radical species.

Mechanisms of arsenic-induced cross-tolerance to nickel cytotoxicity, genotoxicity, and apoptosis in rat liver epithelial cells.

The purpose of the present study was to investigate the mechanism of cross-tolerance to nickel in arsenic-transformed cells. Chronic arsenite-exposed (CAsE) cells (TRL 1215 cells, which had been continuously exposed to 0.5 microM arsenite for 20 or more weeks) and control TRL 1215 cells were both exposed to nickel for 24 h, and cell viability was determined by metabolic integrity. The LC(50) for nickel was 608 +/- 32 microM in CAsE cells as compared to 232 +/- 16 microM in control cells, a 2.6-fold increase. CAsE and control cells were treated with 200 microM nickel for 4 h and cellular-free radical production was measured using ESR spectrometry. Hydroxyl radical generation was decreased in CAsE cells. Thiobarbituric acid reactive substances, indicative of lipid peroxidation, and 8-oxo-2'-deoxyguanosine, indicative of oxidative DNA damage, were reduced in CAsE cells. Flow cytometric analysis using Annexin/FITC revealed that nickel-induced apoptosis was reduced in CAsE cells. CAsE cells showed generalized resistance to oxidant-induced toxicity as evidenced by a marked reduction in sensitivity to hydrogen peroxide. Interestingly, intracellular reduced glutathione (GSH) levels were significantly increased in CAsE cells, and when GSH was depleted, CAsE cells lost their nickel resistance. The mechanism of arsenic-induced cross-tolerance to cytotoxicity, genotoxicity, and apoptosis induced by nickel appears related to a generalized resistance to oxidant-induced injury, probably based, at least in part, in increased cellular GSH levels.

Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus.

Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H. hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N(2) malondialdehyde-deoxyguanosine (M1dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M1dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M1dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 10(8) nucleotides, respectively. Higher levels of M1dG were detected in the liver DNA of H. hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 10(8) nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M1dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M1dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H. hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M1dG occurs as a result of oxidative stress associated with H. hepaticus infection of mice, and may contribute to liver carcinogenesis in this model.

Presence of potential nickel-responsive element(s) in the mouse MTH1 promoter.

The murine MTH1 gene codes for MTH1, an 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) which hydrolyzes 8-oxo-dGTP, a promutagenic product of reactive oxygen species' attack on the nucleotide pool. This gene is regulated by oxidative stress. Therefore, we hypothesized that MTH1 expression can be affected by carcinogenic nickel(II), known to induce such stress. Three plasmid constructs, carrying different upstream regions of the mouse MTH1 and the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently transfected into NIH 3T3 cells and the CAT protein was measured in nickel(II) acetate-treated and untreated cells. Nickel concentration-dependent increase of CAT protein level was observed for low Ni(II) concentrations, up to 400 microM Ni(II), in cells transfected with pHI103 plasmid (-5969 to +530 of the MTH1 sequence) only. Cells transfected with the pHI104 (-1331 to +530) or pHI108 (-151 to +530) plasmids did not respond to nickel(II) whatsoever. This finding demonstrated that the MTH1 sequence between -5969 and -1331 contained element(s) responsive to nickel(II) treatment. DNA sequencing revealed the presence of AP-1, NF-kappaB, and ATF-1 binding sites in both the -5969 to -1331 and -1331 to +530 regions. In contrast, two (CA)n repeats (-5642 to -5582 and -2078 to -2031), a family of B2 (-5428 to -5247) and B1 (-4559 to -4420) short interspersed repeated elements, and an (AT)n repeat (-5243 to -5230) were identified only in the -5969 to -1331 sequence. The results suggest that up-regulation of murine MTH1 expression by nickel(II) is controlled by the repeat sequences, potential candidates for nickel-responsive elements.

K-ras mutations in mouse lung tumors of extreme age: independent of paternal preconceptional exposure to chromium(III) but significantly more frequent in carcinomas than adenomas.

Preconceptional exposure of male NIH Swiss mice to chromium(III) chloride resulted in increased incidence of neoplastic and non-neoplastic changes in their progeny, including lung tumors in females [Toxicol. Appl. Pharmacol. 158 (1999) 161-176]. Since mutations in the K-ras protooncogene are frequent, early changes in mouse lung tumors, we investigated possible mutational activation of this gene as a mechanism for preconceptional carcinogenesis by chromium(III). These offspring had lived until natural death at advanced ages (average 816+/-175 days for controls, 904+/-164 for progeny of chromium-treated fathers). Mutations of K-ras, analyzed by single-strand conformation polymorphism and sequencing, were, in codon 12, wild type GGT (glycine), to GAT (aspartic acid); to GTT (valine); and to CGT (arginine); and in codon 61, wild-type CAA (glutamine), to CGA (arginine). K-ras mutation frequencies in lung tumors were very similar in control progeny (4/14) and in progeny of chromium-treated fathers (5/15). Thus, germline mutation or tendency to spontaneous mutation in K-ras does not seem to be part of the mechanism of preconceptional carcinogenesis here. However, an additional interesting observation was that K-ras mutations were much more frequent in lung carcinomas (8/16) than in adenomas (1/13) (P=0.02), for all progeny combined. This was not related to age of the tumor-bearing mice or the size of the tumors. K-ras mutations may contribute to malignant tumor progression during aging, of possible relevance to the putative association of such mutations with poor prognosis of human lung adenocarcinomas.

Intracellular distribution of the antimutagenic enzyme MTH1 in the liver, kidney and testis of F344 rats and its modulation by cadmium.

Cellular distribution of the antimutagenic MTH1protein in the liver, kidney, and testis of Fischer rat was evaluated using the immunohistochemical staining with anti-MTH1 polyclonal antibody. The present investigation revealed a non-uniform distribution of MTH1 among cells and among the cytoplasmic, nuclear, and membranal structures of cells within a given tissue. A particularly strong expression of MTH1 was observed for the first time in the perinuclear acrosomic bodies of spermatocytes and in the acrosomic vesicles of sperm heads. Treatment of rats with a single sc dose of 20 micromol Cd(II)/kg body wt. produced histopathologic changes in these organs accompanied by redistribution of the cellular MTH1 protein between the cytoplasm and nuclei. The acute phase of Cd(II) toxicity, that in the liver and especially in the testes (but not in kidneys) led to cell necrosis, was accompanied by a characteristic decrease in the abundance of MTH1-expressing nuclei. Chronic toxicity without necrosis, persisting in the kidney over the entire 14-day study, as well as the survival and proliferation of cells, observed in the liver and testis after the necrotizing phase, were signified by increased number of nuclei expressing MTH1. Thus, unlike previous biochemical studies, immunohistochemistry managed to reveal alterations in the patterns of inter- and intracellular distribution of MTH1, associated apparently with the conditional changes in the dynamics of synthesis of nucleic acids, assisted by this protein.

Induction of a secondary structure in the N-terminal pentadecapeptide of human protamine HP2 through Ni(II) coordination. An NMR study.

A solution structure of the Ni(II) complex with the N-terminal pentadecapeptide of human protamine HP2 (HP2(1)(-)(15)) was elucidated with the use of a range of one- and two-dimensional (1)H NMR techniques and molecular modeling. A striking double-loop conformation was found, exhibiting the interactions of the aromatic ring of the Tyr(8) residue with the Ni(II) coordination site at Arg(1), Thr(2), and His(3) residues and the side chain of the Arg(15) residue. In such a conformation, a tendency was found for all five positively charged arginine side chains to locate on one side of the molecule, making possible efficient contacts with the DNA double helix. These structural features, induced indirectly by Ni(II) coordination, are discussed in terms of a possible physiological function of the N-terminus of HP2 as a metal-binding site.

Possible roles of nitric oxide and redox cell signaling in metal-induced toxicity and carcinogenesis: a review.

Toxic doses of transition metals are capable of disturbing the natural oxidation/reduction balance in cells through various mechanisms stemming from their own complex redox reactions with endogenous oxidants and effects on cellular antioxidant systems. The resulting oxidative stress may damage redox-sensitive signaling molecules, such as NO, S-nitrosothiols, AP-1, NF-kappaB, IkappaB, p53, p21ras, and others, and thus derange the cell signaling and gene expression systems. This, in turn, may produce a variety of toxic effects, including carcinogenesis. Experimental support for the relevance of oxidative damage to the mechanisms of metal toxicity and carcinogenicity is particularly strong for two essential (but toxic when overdosed) metals--iron and copper-- and three well-established human metal carcinogens--nickel, chromium, and cadmium. However, along with more specific effects of toxic metals associated with their selective binding to particular cell constituents and affecting calcium signaling, oxidative damage seems to become important as well in explaining mechanisms of pathogenicity of other metals, such as lead, mercury, and arsenic.

Ni(II) specifically cleaves the C-terminal tail of the major variant of histone H2A and forms an oxidative damage-mediating complex with the cleaved-off octapeptide.

The acetyl-TESHHK-amide peptide, modeling a part of the C-terminal "tail" of histone H2A, was found previously by us to undergo at pH 7. 4 a Ni(II)-assisted hydrolysis of the E-S peptide bond with formation of a stronger Ni(II) complex with the SHHK-amide product [Bal, W., et al. (1998) Chem. Res. Toxicol. 11, 1014-1023]. To further characterize the hydrolysis and test the resulting Ni(II) complex for redox activity, bovine histone H2A and three peptides were investigated: acetyl-LLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK (H2A(34)), modeling the entire "C-tail" of H2A; SHHKAKGK (H2A(8)), modeling the cutoff product of hydrolysis; and acetyl-KTESHKAKGK (H2A(10)), modeling a putative Ni(II) binding site in a minor variant H2A.4 of human histone H2A. The Ni(II)-assisted hydrolysis of H2A and H2A(34) was found to proceed approximately 7-fold faster than that of the Ni(II)-acetyl-TESHHK-amide complex under comparable conditions. In both cases, the Ni(II) complex with H2A(8) was the smaller product of the hydrolysis, indicating a high site specificity of the reaction. Of three other metals tested with H2A(34), only Cu(II) cleaved the E-S bond, although much less efficiently than Ni(II); Co(II) and Zn(II) had no effect whatsoever. The H2A(10) peptide appeared to be fully resistant to hydrolytic cleavage and did not exhibit any redox activity versus H(2)O(2) in the presence of Ni(II) at pH 7.4. Likewise, redox-inactive was the Ni(II)-H2A(34) complex. In contrast, the Ni(II)-H2A(8) complex promoted oxidative damage of pUC19 DNA by H(2)O(2), evidenced by a significant increase in the number of single strand breaks and nucleobase modifications typical for a hydroxyl radical-like species attack on DNA. Interestingly, instead of 8-oxopurines, the corresponding formamidopyrimidines were the major products of the damage. The difference in redox activity between the Ni(II)-H2A(34) and Ni(II)-H2A(8) complexes is most likely associated with their different geometries: octahedral and square planar, respectively. Incubation of the Ni(II)-H2A(8) complex with H(2)O(2) also resulted in degradation of the peptide ligand, especially at its Ser and His residues. Thus, binding of Ni(II) to the ESHHK motif of the histone H2A C-tail is damaging to the histone C-terminal tail and to histone-associated DNA. The results support a dual mechanism of Ni(II)-induced carcinogenesis, including both genotoxic and epigenetic effects.

Molecular models in nickel carcinogenesis.

Nickel compounds are known human carcinogens, but the exact molecular mechanisms of nickel carcinogenesis are not known. Due to their abundance, histones are likely targets for Ni(II) ions among nuclear macromolecules. This paper reviews our recent studies of peptide and protein models of Ni(II) binding to histones. The results allowed us to propose several mechanisms of Ni(II)-inflicted damage, including nucleobase oxidation and sequence-specific histone hydrolysis. Quantitative estimations of Ni(II) speciation, based on these studies, support the likelihood of Ni(II) binding to histones in vivo, and the protective role of high levels of glutathione. These calculations indicate the importance of histidine in the intracellular Ni(II) speciation.

Inhibition of antimutagenic enzymes, 8-oxo-dGTPases, by carcinogenic metals. Recent developments.

Nickel, cadmium, cobalt, and copper are carcinogenic to humans and/or animals, but the underlying mechanisms are poorly understood. Our studies have been focused on one such mechanism involving mediation by the metals of promutagenic oxidative damage to DNA bases. The damage may be inflicted directly in DNA or in the deoxynucleotide pool, from which the damaged bases are incorporated into DNA. Such incorporation is prevented in cells by 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphatases (8-oxo-dGTPases). Thus, inhibition of these enzymes should enhance carcinogenesis. We have studied effects of Cd(II), Cu(II), Co(II), and Ni(II) on the activity of isolated bacterial and human 8-oxo-dGTPases. Cd(II) and Cu(II) were strongly inhibitory, while Ni(II) and Co(II) were much less suppressive. After developing an assay for 8-oxo-dGTPase activity, we confirmed the inhibition by Cd(II) in cultured cells and in the rat testis, the target organ for cadmium carcinogenesis. 8-Oxo-dGTPase inhibition was accompanied by an increase in the 8-oxo-dG level in testicular DNA.

Oxidative DNA damage in fetal tissues after transplacental exposure to 3'-azido-3'-deoxythymidine (AZT).

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) has been used successfully to reduce the incidence of transplacental and perinatal transmission of the HIV virus. However, prolonged treatment with high doses of AZT is utilized in this therapy, and AZT has been found to be a perinatal carcinogen in mice. Any possible perinatal carcinogenic side effects in the human can best be managed if the mechanism is understood. AZT targets mitochondria and might cause increased intracellular production of reactive oxygen species (ROS). We tested whether transplacental AZT may cause oxidative damage in nuclear DNA of fetal tissues. CD-1 Swiss pregnant mice were treated with the transplacental carcinogenesis regimen (25 mg/day AZT, for gestation days 12-18) and tissues collected on the day of birth. Significant increases in 8-oxo-2'-deoxyguano- sine (8-oxo-dG) were found in the livers, a target tissue for transplacental carcinogenesis, and in the kidneys. A non-significant increase occurred in brain, with no change in lung. Tissues were also obtained from fetal patas monkeys (Erythrocebus patas), whose mothers had received 10 mg AZT/day during the last half of gestation. Although limited numbers of samples were available, possible increases in 8-oxo-dG were noted, relative to controls, for placenta and for fetal lung and brain (P = 0.055 for treatment-related increases in these tissues). These results suggest that an increase in reactive oxygen species could contribute to the mechanism of transplacental carcinogenesis by AZT in mice, and that this may also occur in primates.

Activity of the antimutagenic enzyme 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) in cultured chinese hamster ovary cells: effects of cell cycle, proliferation rate, and population density.

Mammalian 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolases (8-oxo-dGTPases), such as MTH1, are believed to play the same antimutagenic role as their bacterial homologues, like MutT. Both decompose promutagenic 8-oxo-dGTP, a product of active oxygen's attack on dGTP. It is not known how 8-oxo-dGTPase expression and function are regulated. Therefore, we investigated the effect of cell population density, proliferation rate, and cell cycle phase on 8-oxo-dGTPase specific activity in cultured Chinese hamster ovary K1-BH4 (CHO) cells. With increasing cell population density (from 30 to 95% confluence), the activity of 8-oxo-dGTPase per milligram protein decreased by 33% (p =.007 by ANOVA) while cells shifted by 9% into the G(0)/G(1) phase, with a 5% drop in cells in S phase. Importantly, inhibition of the cells' proliferation rate by calf serum deprivation caused a more dramatic 23% shift toward the G(0)/G(1) phase and a 25% drop in S phase, but had no effect on 8-oxo-dGTPase activity. Likewise, no differences in the enzyme activity were observed within cell populations of different cell cycle phases separated by centrifugal elutriation. Thus, the present results exclude cell cycle-dependent regulation of 8-oxo-dGTPase activity in CHO cells or its simple dependence on proliferation rate. The observed decrease of 8-oxo-dGTPase activity with increasing cell population density might be related to augmentation of cell-to-cell contact.

Oxidative DNA damage in liver of mice with different susceptibility to hepatocarcinogenesis.

Susceptibility to spontaneous or chemically-induced liver tumors in mice has been demonstrated to be strain dependent, with the tumor development or promotion phase contributing most to variability. Since reactive oxygen species are thought to play a role in carcinogenesis, especially tumor promotion, we investigated steady-state levels of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in hepatic DNA from age matched (7 months), untreated mice of three inbred strains, that differ significantly in their susceptibility to liver carcinogenesis. Male mice of strain C3H, highly sensitive to liver tumorigenesis, had significantly higher levels of hepatic 8-oxo-dG (3.3 SE 0.2 8-oxo-dG/10(5) dG) than resistant strains C57BL (2.1 SE 0.3/10(5) dG; p < 0.01) and A/JCr (2.5 SE 0.1/10(5) dG; p < 0.015). In contrast, levels of 8-oxo-dG in livers of female A/JCr mice (3.1 SE 0.3/10(5) dG) were higher than in those of C3H (2.2 SE 0.1/10(5) dG; p < 0.025) and C57BL females (2.5 SE 0.3/10(5) dG; p = NS). Male C3H livers presented significantly more 8-oxo-dG than those of C3H females (p < 0.002). These results suggest that steady-state levels of 8-oxo-dG may contribute to the inherent differences in susceptibility to hepatocarcinogenesis in inbred strains of mice, especially the high sensitivity of C3H males.

Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs.

Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA. One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA. During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8). The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above). Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells.

Cadmium(II), unlike nickel(II), inhibits 8-oxo-dGTPase activity and increases 8-oxo-dG level in DNA of the rat testis, a target organ for cadmium(II) carcinogenesis.

8-Oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) is an enzyme which prevents incorporation into DNA of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) from a deoxynucleotide pool damaged by endogenous oxidants. Its inhibition may thus be carcinogenic. We previously found that Cd(II) inhibited 8-oxo-dGTPase in both cell free systems and cultured cells. To verify this finding in a relevant animal model, we investigated the effects of Cd(II) on cellular 8-oxo-dGTPase activity and nuclear DNA 8-oxo-dG levels in the rat testis, a target organ for Cd(II) carcinogenesis. Ni(II), which does not induce testicular tumors in rats and is a weaker in vitro inhibitor of 8-oxo-dGTPase than Cd(II), was investigated as a comparison. Male F344/NCr rats were given a single s.c. dose of 20 micromol Cd(II) acetate, 90 micromol Ni(II) acetate or 180 micromol sodium acetate (controls) per kg body wt and killed 2, 8, 24 or 48 h later (three rats/time point). Cd(II) caused a gradual decrease in testicular 8-oxo-dGTPase activity with time. It became significant only after 8 h post-injection (P < 0.05) and resulted in a final 50% loss of the enzyme activity at 48 h (P < 0. 01). Although the results for Ni(II) at 8 h and later were apparently lower than the controls, the decrease did not reach statistical significance. Treatment of rats with Cd(II) led to an early and progressive increase (from 130% at 2 h to 200% at 48 h versus the controls) of the 8-oxo-dG level in testicular DNA (P < 0. 05 or better). Ni(II) acetate also tended to raise the testicular 8-oxo-dG level, but the increase was transient, with an apparent maximum at 8 h, and did not approach statistical significance (P < 0. 2). Thus, Cd(II), unlike Ni(II), is able to inhibit 8-oxo-dGTPase activity and to raise 8-oxo-dG levels in rat testicular DNA. However, the time course of both effects indicates that 8-oxo-dGTPase inhibition is most likely not the sole cause of the increase in 8-oxo-dG.

Preconception urethane or chromium(III) treatment of male mice: multiple neoplastic and non-neoplastic changes in offspring.

Increase in neoplasia in offspring after preconception exposure of parents presents puzzling features such as high frequency of effects and lack of Mendelian inheritance. The present study examined the hypothesis that preconception carcinogenesis involves an increase in the rate of occurrence of neoplasms with a spontaneous incidence. Male NIH Swiss mice (12 per group) were exposed 2 weeks before mating (once, ip) to urethane (1.5 g/kg) or chromium(III) chloride (1 mmol/kg). Offspring (48-78/sex/group) were examined for all grossly apparent changes when moribund or at natural death, followed by histopathological diagnosis and statistical analysis. Significant exposure-related changes occurred in multiple organs. Ten to 20 percent of offspring showed changes related to paternal exposure, including at least one sired by most treated males. Pheochromocytomas occurred in both male and female offspring after both treatments, with none in controls. These neoplasms are rare in mice and suggest endocrine dysfunction as a component of preconception carcinogenesis. This was supported by increases in thyroid follicular cell and Harderian gland tumors, ovarian cysts, and uterine abnormalities. Lung tumors were increased in female offspring only. Effects seen in offspring only after paternal urethane exposure were an increase in preneoplasia/neoplasia in the glandular stomach (males) and in females, increased lymphoma but decreased incidence of histiocytic sarcoma. Increases in incidence of male reproductive gland tumors and of renal non-neoplastic lesions occurred only after chromium exposure. Thus, preconception exposure of fathers to toxicants had a significant impact on both neoplastic and non-neoplastic changes in almost all tissues in which these lesions often occur naturally during the aging process.

Nickel(II) acetate-treated Chinese hamster ovary cells differentially express Vimentin, hSNF2H homologue, and H ferritin.

In probing the possible non-genotoxic molecular mechanism(s) of nickel(II)-induced carcinogenesis, we performed a non-radioactive mRNA differential display analysis for nickel(II) acetate-treated Chinese hamster ovary cells (CHO-K1-BH4). Three out of thirty differentially expressed cDNAs had sequences highly similar to known genes. Down-regulation of vimentin and a hSNF2H homologue and up-regulation of ferritin heavy chain were confirmed by Northern blot analysis. The expression of these mRNAs was time- and nickel(II) concentration-dependent. For vimentin, the decrease in mRNA level was concurrent with a decrease in the protein level. For ferritin, the increase in mRNA had no effect on the protein level. Dysregulation of these gene products signifies their involvement in the epigenetic effects of carcinogenic nickel(II) compounds.