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.

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.

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.

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.

Involvement of K+ATP channels in nitric oxide-induced inhibition of spontaneous contractile activity of the nonpregnant human myometrium.

Nitric oxide (NO), an important endogenous substance, is known to be a strong relaxant of smooth muscle, including myometrium. It has been postulated that the relaxing effect of NO on smooth muscle is achieved by the stimulation of soluble guanylyl cyclase, which leads to an increase in the cyclic guanosine 3',5'-monophosphate (cGMP) levels and hyperpolarization of the cellular membrane. The aim of our study was to investigate the involvement of K+ATP channels in the mechanism of cGMP-independent nitric oxide-induced inhibition of contractile activity of the nonpregnant human myometrium, obtained at hysterectomy. Nitric oxide's influence on contractile activity was recorded in the presence of methylene blue and glybenclamide, blockers of soluble guanylyl cyclase and K+ATP channels, respectively. Nitric oxide, generated by the NO donor DEA/NO, caused a dose-dependent inhibition of the spontaneous contractile activity of human nonpregnant myometrium. Preincubation with methylene blue (5 microM) did not prevent NO-induced relaxation of uterine strips, while 1.5 microM glybenclamide blocked this effect. Our results indicate that nitric oxide relaxes human non-pregnant uterus through K+ATP channels, independent of the cGMP pathway.

Distinct time courses of increase in cytochromes P450 1A2, 2A5 and glutathione S-transferases during the progressive hepatitis associated with Helicobacter hepaticus.

Mice naturally infected by Helicobacter hepaticus develop a chronic active hepatitis leading to hepatocellular carcinoma. This mouse model of liver cancer was used to examine the impact of bacterial infection on the hepatic expression and activity of enzymes involved in carcinogen bioactivation (phase I enzymes) and detoxification (phase II enzymes). No major differences in total cytochrome P450 (CYP) content were found between control and infected mice during the course of the study. The most striking modulations of individual isoenzymes were the increases in immunohistochemical staining observed for CYP1A and CYP2A5 in relation to increasing age and liver lesions. The increase in CYP2A5 in mice aged over 12 months was confirmed by the observed increases in coumarin 7-hydroxylation (CYP2A5 substrate) in vitro and CYP2A5 mRNA levels by Northern blot analysis. Immunoblotting confirmed the specific induction of CYP1A2 in infected mice 12 and 18 months of age. Perfusion of liver with nitroblue tetrazolium, an indicator for superoxide formation, demonstrated that in livers of infected mice, hepatocytes often co-expressed CYP2A5 and formazan deposition. Concerning phase II enzymes, an enhancement of glutathione S-transferase (GST) activities, related to the disease process, was observed in infected mice. An age-specific increase of GSTpi and A4.4 (early stage of disease) and GST YaYa (>9 months) expression was also demonstrated by immunohistochemical staining. In contrast, catalase and glutathione-peroxidase activities, as well as reduced glutathione content were decreased in the early stages of disease (3-9 months) in infected mice compared to age-matched control mice. Overall, these results suggest that alterations in CYP and GST expression may contribute to the aetiology of tumour incidence due to H. hepaticus infection via production of reactive oxygen species.

Increased oxidative DNA damage and hepatocyte overexpression of specific cytochrome P450 isoforms in hepatitis of mice infected with Helicobacter hepaticus.

A recently discovered bacterium, Helicobacter hepaticus, infects the intrahepatic bile canaliculi of mice, causing a severe chronic hepatitis culminating in liver cancer. Thus, it affords an animal model for study of bacteria-associated tumorigenesis including H. pylori-related gastric cancer. Reactive oxygen species are often postulated to contribute to this process. We now report that hepatitis of male mice infected with H. hepaticus show significant increases in the oxidatively damaged DNA deoxynucleoside 8-hydroxydeoxyguanosine, with the degree of damage increasing with progression of the disease. Perfusion of infected livers with nitro blue tetrazolium revealed that superoxide was produced in the cytoplasm of hepatocytes, especially in association with plasmacytic infiltrates near portal triads. Contrary to expectations, Kupffer cells, macrophages, and neutrophils were rarely involved. However, levels of cytochrome P450 (CYP) isoforms 1A2 and 2A5 in hepatocytes appeared to be greatly increased, as indicated by the number of cells positive in immunohistochemistry and the intensity of staining in many cells, concomitant with severe hepatitis. The CYP2A5 immunohistochemical staining co-localized with formazan deposits resulting from nitro blue tetrazolium reduction and occurred in nuclei as well as cytoplasm. These findings suggest that CYP2A5 contributes to the superoxide production and 8-hydroxydeoxyguanosine formation, although reactive oxygen species from an unknown source in the hepatocytes leading to CYP2A5 induction or coincidental occurrence of these events are also possibilities. Three glutathione S-transferase isoforms, mGSTP1-1 (pi), mGSTA1-1 (YaYa), and mGSTA4-4, also showed striking increases evidencing major oxidative stress in these livers.

Oxidative DNA damage in tissues of pregnant female mice and fetuses caused by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induces the promutagenic oxidative-damage DNA lesion, 8-oxo-2'-deoxyguanosine (8-oxo-dG), in adult animals. To investigate whether this alteration occurs in DNA after transplacental exposure, pregnant Swiss mice were administered single or multiple doses of NNK. The 8-oxo-dG was quantified in placenta, and maternal and fetal tissues. In maternal lungs, single and multiple doses of NNK significantly increased levels of 8-oxo-dG by 23% and 32%, respectively. In maternal liver, a significant 38% increase was observed after multiple dose treatment. In the fetuses, a significant 45% increase in 8-oxo-dG levels was observed in liver after multiple doses of NNK. This is the first demonstration of oxidative DNA damage after transplacental exposure to NNK, and supports the concept of maternal smoking as a contributor to the development of childhood cancer.

Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H. hepaticus infection causes liver cancer through other pathways than direct damage to DNA.

Uptake and tissue distribution of chromium(III) in mice after a single intraperitoneal or subcutaneous administration.

The tissue levels of chromium were followed after single intraperitoneal or subcutaneous injection of 1 mmol CrCl3/kg body wt. in Swiss male mice. Blood levels were similar after both treatment modes, with half-lives of 31-41 h. Organs not directly exposed by i.p. treatment contained similar amounts in the two groups, with kidneys > lungs > heart > brain. However, after i.p. treatment peritoneal organs (liver, spleen, pancreas and testis) had 40- to 200-fold more chromium compared with s.c. Assay of subsurface liver tissue and of testes removed via the scrotum indicated infiltration of the organs, rather than surface adsorption, of peritoneal chromium. Relative chromium concentrations after i.p. treatment were liver > pancreas = spleen > testis and after s.c. liver > spleen > testis > pancreas. Thus, s.c. treatment with CrCl3 is as effective as i.p. in terms of absorption into the blood. Treatment i.p., leading to direct uptake into peritoneal organs, is an effective way to deliver high chromium doses to these organs, but does not model likely human exposure.

Liver tumorigenesis by Helicobacter hepaticus: considerations of mechanism.

A new animal model for the causation of liver tumors via a bacterial infection presented itself fortuitously in the form of a new species, Helicobacter hepaticus. This species of Helicobacter colonizes the hepatic bile canaliculi in susceptible strains of mice, resulting in hepatitis and hepatocellular and hepatocholangiolar adenomas and carcinomas. The mechanism by which this infection leads to cancer is unknown. Tests with Helicobacter hepaticus have revealed thus far that the bacteria do not secrete a mutagen which is capable of detection by the Ames Assay. Measurement of oxidatively damaged bases in the liver DNA of hepaticus infected mice have shown accumulation of 8-oxodeoxyguanosine with disease progression. Other promutagenic DNA lesions, 7-methylguanine and O6-methylguanine, indicative of nitrosation of endogenous amines by nitric oxide, were not detected. Analysis of carcinomas and adenomas taken from H. hepaticus infected A/JCr mice revealed no mutations in ras oncogenes or in exons 5-8 of the p53 gene. These preliminary results indicate that a non-genotoxic tumor promotion mechanism, possibly implemented by reactive oxygen species from the immune response, is more likely than a genotoxic mechanism.