Asbestos catalyzes the formation of the 6-oxobenzo[a]pyrene radical from 6-hydroxybenzo[a]pyrene

Crocidolite asbestos catalyzes the oxidation of 6-hydroxybenzo[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene radical as determined by electron spin resonance spectroscopy. This may be a mechanism whereby inhaled asbestos enhances the incidence of lung cancer induced by cigarette smoke, which contains benzo[a]pyrene.     

Ankylosis of hock joints in group caged male B6C3F1 mice

Enlarged hock joints were observed during 1983 in B6C3F1 mice of chronic toxicity and carcinogenicity studies sponsored by the National Toxicology Program (NTP). Subsequently, approximately 9,500 B6C3F1 mice on 32 NTP chemical toxicity and carcinogenicity studies were evaluated for this condition by clinical examination. Group caged male B6C3F1 mice had thickening and reduced mobility of the hock joints at prevalences of 1.2% up to 6 months of age; 23% at 6 to 12 months of age; and 62% at 13 to 26 months of age. Group caged female B6C3F1 mice had a prevalence of 2% or less. Histologically, affected mice had periarticular exostoses on the bones of the hock joints, with formation of bony bridges around joints and deposition of new bone in joint spaces, resulting in partial or complete ankylosis. Individually caged male and female B6C3F1 mice were not affected. The cause of the ankylosis was not determined, but its occurrence in the NTP studies has been reduced by individual caging.     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

Hepatic DNA and RNA adduct formation from the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene and its electrophilic sulfuric acid ester metabolite in preweanling rats and mice

DNA and RNA adducts that were chromatographically identical to those formed in vitro on reaction of 7-sulfooxymethyl-12-methyl-benz[a]anthracene with guanine and adenine nucleosides were formed in the livers of rats and mice given i.p. injections of 7-hydroxymethyl- or 7-sulfooxymethyl-12-methyl-benz[a]anthracene. Considerably higher levels of these hepatic adducts were obtained from the latter short-lived electrophilic ester than from the hydroxymethyl compound. These observations are consistent with the finding of rat liver cytosolic sulfotransferase activity for 7-hydroxymethyl-12-methylbenz[a]anthracene (Watabe et al., Science 215, 403, 1982). Formation of these hepatic adducts from 7-hydroxymethyl-12-methylbenz[a]anthracene was inhibited by prior administration to rats of dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for this hydroxymethyl hydrocarbon.     

Diminished energy metabolism and enhanced apoptosis in livers of B6C3F1 mice treated with the antihepatocarcinogen rotenone

Rotenone decreases the incidence of hepatocellular carcinoma and lowers rates of hepatocellular proliferation. In an effort to delineate mechanisms involved, the in vivo effect of rotenone on liver mitochondrial metabolism, apoptotic machinery as well as elements of the hepatic signal transduction pathways were investigated. Mitochondria from livers of male B6C3F1 mice fed a standard diet containing 600 ppm rotenone for 7 days were uncoupled or inhibited when succinate or glutamate plus malate were used as the substrate, respectively. These livers also showed a significant increase in apoptosis compared with control livers. Furthermore, rotenone increased the expression of c-myc mRNA to 5-fold of control values within 3 days, an effect which was still observed (3-fold) after 7 days. Levels of p53 mRNA were also increased 3-fold after 1 day, but declined to control levels by 7 days. Rotenone also caused a transient, yet marked increase in liver particulate glyceraldehyde phosphate dehydrogenase (GAPDH) protein expression, while it did not alter the expression of the cytosolic form of the enzyme. Conversely, mRNA of the proto-oncogene H-ras showed a decline of 35% after 3 days of rotenone treatment, and remained diminished for the duration of the experiment. These data suggest that rotenone may act as an anticancer agent by diminishing mitochondrial bioenergetics which prevents basal hepatocyte proliferation and lowers the threshold for liver cells with DNA damage to undergo apoptosis.     

Benzene increases protein-bound 3-nitrotyrosine in bone marrow of B6C3F1 mice

Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.     

Incidence of obstructive uropathy in male B6C3F1 mice on a 24-month carcinogenicity study and its apparent prevention by ochratoxin A

A 24-month study assessed the carcinogenic potential of the nephrotoxic mycotoxin ochratoxin A (OA) in B6C3F1 mice. Three groups of 50 males and 50 females were fed 0.1 or 40 ppm OA in the diet. Obstructive urinary tract disease (mouse urological syndrome [MUS]) accounted for the greatest number of spontaneous deaths in the male mice of control (12/50) and 1 ppm (13/50) dose groups, but the disease was not observed in the males fed 40 ppm OA. The earliest age of onset of clinical signs of MUS was 4 months and the average age of onset was 10.1 months. The first death from MUS was observed at 5 months and average age at death was 12.2 months. The mice were caged in groups of five mice per cage and clustering of cases of MUS was observed. Properties of OA which may be important to its preventive effect include inhibition of growth of gram positive bacteria and the production of polyuria as a result of renal proximal tubular damage.     

The role of Klebsiella oxytoca in utero-ovarian infection of B6C3F1 mice

A disease consisting of suppurative endometritis, salpingitis, perioophoritis and/or peritonitis has been an important problem in aging B6C3F1 mice on some chronic chemical carcinogenicity studies. Klebsiella oxytoca was identified as the most likely causative agent based on cultural isolations from lesions. A study was done to determine prevalence of K. oxytoca in the "normal" flora of mice from different breeding facilities. In a survey of 684 retired female breeder mice from 10 National Institutes of Environmental Health Sciences (NIEHS) and National Cancer Institute (NCI) production facilities, K. oxytoca was isolated from only 1% of nasopharynxes, vaginas and ceca in mice from 7 of 10 facilities. Epizootiology of the natural infection was investigated using the capsular and biochemical typing methods on 97 isolates of K. oxytoca from mice of 11 NIEHS and NCI production facilities and sentinel mice from three National Toxicology Program testing facilities. A few capsular types were associated with either lesions, nonlesion isolation sites, or certain facilities but the capsular typing method was not reproducible. No associations were found for any biotypes. A K. oxytoca isolate (capsular type 20, biotype A) from a typical case of perioophoritis was used in attempts to reproduce the natural disease in Klebsiella-free B6C3F1 female mice. Mice were inoculated at 6 months of age by the intravaginal, intrauterine or intraperitoneal route with one of four doses of K. oxytoca and killed at 4, 7 or 10 months post-infection. Some mice given high doses (10(6) or 10(8) colony forming units) of K. +oxytoca died of septicemia and a few developed mild inflammatory lesions in the uterus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of trichloroethylene on DNA methylation and expression of early-intermediate protooncogenes in the liver of B6C3F1 mice.

Trichloroethylene (TCE) is a multimedia environmental pollution that is carcinogenic in mouse liver. The ability of TCE to modulate DNA methylation and the expression of immediate-early protooncogenes was evaluated. Female B6C3F1 mice were administered 1000 mg/kg TCE by gavage 5 days/week and killed after 5, 12, or 33 days of exposure. Methylation of DNA as 5-methylcytosine was decreased by 5 days of treatment with TCE and remained reduced for 33 days. TCE also decreased the methylation of the promoter regions for the protooncogenes, c-jun and c-myc. The expression of the mRNA for the two protooncogenes was increased between 60 and 120 minutes after administering the last dose of TCE and returned to control level by 24 hours. The expression of the mRNA for c-fos remained undetectable after administering TCE. Hence, TCE decreased the methylation both of total DNA and the promoters for the c-jun and c-myc genes and increased the expression of their mRNA. The decreased methylation and increased expression of the two immediate-early protooncogenes might be associated with TCE-induced increase in cell proliferation and promotion of tumors.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as “maters”), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB₁) were orchidectomized and identified as either maters or “non-maters”. A significant proportion of the BXB₁ maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB₁ maters relative to the B6D2F1 and BXB₁ non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior.     

The effect of dichloroacetic acid and trichloroacetic acid on DNA methylation and cell proliferation in B6C3F1 mice

The chlorine disinfection by-products, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are carcinogenic in mouse liver. We have previously reported that DCA and TCA induced DNA hypomethylation in mouse liver. In the present study, we determined the temporal association for DNA hypomethylation and cell proliferation. Female B6C3F1 mice were administered daily doses of 500 mg/kg DCA or TCA by gavage and sacrificed at 24, 36, 48, 72, and 96 hours after the first dose. The proliferating cell nuclear antigen-labeling index in the liver was increased at 72 and 96 hours by both DCA and TCA, that is, at 72 hours the index was 1.00 +/- 0.21, 0.51 +/- 0.11, and 0.095 +/- 0.016 for DCA, TCA, and the vehicle control, respectively. The mitotic index was also significantly increased at 96 hours. The promoter region for the c-myc gene was hypomethylated only at 72 and 96 hours and not at the earlier sacrifices. Similarly, the methylation of the c-myc gene in the kidney and urinary bladder was decreased only at 72 and 96 hours. In summary, enhancement of cell proliferation and decreased methylation of the c-myc gene were first observed simultaneously at 72 hours after the start of exposure. Thus, the results support the hypothesis that DCA and TCA induce DNA hypomethylation by inducing DNA replication and preventing the methylation of the newly synthesized strands of DNA.     

Carcinogenicity study of GSM and DCS wireless communication signals in B6C3F1 mice

The purpose of this study using a total of 1170 B6C3F1 mice was to detect and evaluate possible carcinogenic effects in mice exposed to radio-frequency-radiation (RFR) from Global System for Mobile Communication (GSM) and Digital Personal Communications System (DCS) handsets as emitted by handsets operating in the center of the communication band, that is, at 902 MHz (GSM) and 1747 MHz (DCS). Restrained mice were exposed for 2 h per day, 5 days per week over a period of 2 years to three different whole-body averaged specific absorption rate (SAR) levels of 0.4, 1.3, 4.0 mW/g bw (SAR), or were sham exposed. Regarding the organ-related tumor incidence, pairwise Fisher's test did not show any significant increase in the incidence of any particular tumor type in the RF exposed groups as compared to the sham exposed group. Interestingly, while the incidences of hepatocellular carcinomas were similar in EMF and sham exposed groups, in both studies the incidences of liver adenomas in males decreased with increasing dose levels; the incidences in the high dose groups were statistically significantly different from those in the sham exposed groups. Comparison to published tumor rates in untreated mice revealed that the observed tumor rates were within the range of historical control data. In conclusion, the present study produced no evidence that the exposure of male and female B6C3F1 mice to wireless GSM and DCS radio frequency signals at a whole body absorption rate of up to 4.0 W/kg resulted in any adverse health effect or had any cumulative influence on the incidence or severity of neoplastic and non-neoplastic background lesions, and thus the study did not provide any evidence of RF possessing a carcinogenic potential.     

The effect of exposure regimen and duration on benzene-induced bone marrow damage in mice. II. Strain comparisons involving B6C3F1, C57B1/6 and DBA/2 male mice

In a companion paper (Luke et al., 1988), the effect of exposure duration and regimen on benzene induced-bone marrow damage was evaluated in male and female DBA/2 mice using the peripheral blood micronucleus assay. To assess the general applicability of the findings obtained for DBA/2 mice to other strains, similar studies were conducted using B6C3F1 and C57B1/6 male mice. An analysis of peripheral blood smears taken weekly from these mice exposed to 300 ppm benzene for 13 weeks (6 h per day) for either 5 days per week (Regimen 1) or for 3 days per week (Regimen 2) revealed: (i) a highly significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE), the magnitude of which was strain specific (DBA/2 greater than C57B1/6 = B6C3F1), but independent of exposure regimen and, except for Regimen 2 B6C3F1 mice, of exposure duration. In male B6C3F1 mice, MN-PCE frequencies increased slightly with increasing exposure duration; (ii) a strain- (C57B1/6 = B6C3F1 greater than DBA/2) and regimen- (Regimen 1 greater than Regimen 2) dependent increase across time in the frequency of micronucleated normochromatic erythrocytes (MN-NCE). Apparent steady-state conditions for MN-NCE frequencies were attained by about 5 weeks of exposure in male mice of all three strains exposed to benzene by Regimen 2. Steady-state conditions for MN-NCE frequencies in male mice exposed to benzene by Regimen 1 did not occur during the duration of the study, with strain-dependent differences in the kinetics of MN-NCE accumulation being present; and (iii) in all 3 strains, an initial severe depression in the rate of erythropoiesis, the return of which to normal levels was both strain- (C57B1/6 = B6C3F1 greater than DBA/2) and regimen- (Regimen 1 greater than Regimen 2) dependent. These data indicate that the induction of genotoxic and cytotoxic damage in the bone marrow of male mice exposed to benzene for 13 weeks can be highly dependent on strain, exposure regimen and exposure duration but that under no circumstance did the level of genotoxic damage induced by benzene decrease under multiple exposure conditions.     

Forestomach ulcers in Crj:B6C3 (C57BL/6NCrj x C3H/HeNCrj) F1 mice

An unusually high incidence of forestomach ulcers was observed in a mouse strain that is used frequently for long-term toxicology studies. Examination of 98 untreated male and 98 untreated female B6C3F1 hybrid mice, the majority of which were between 105 and 113 weeks of age, revealed forestomach ulcers in 52% of the males and 54% of the females. Glandular stomach ulcers were uncommon, being found in only four female mice. The incidence of the ulcers increased with age. The etiology of the lesion is unknown.     

DNA adducts in rats and mice following exposure to [4-14C]-1,2-epoxy-3-butene and to [2,3-14C]-1,3-butadiene

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible than rats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver and lungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative and quantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by comparison with the products formed from the reaction of the individual epoxides with 2'-deoxyguanosine (dG). In rats and mice exposed to [4-14C]-BMO (1-50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung with N7-(2-hydroxy-3-butenyl)guanine (G1) and N7-(1-(hydroxymethyl)-2-propenyl)guanine (G2) as major adducts and N7-2,3,4-trihydroxybutylguanine (G4) as minor adduct. In rats and mice exposed to 200 ppm [2,3-14C]-BD by nose-only inhalation for 6 h, G4 was the major adduct in liver, lung and testes while G1 and G2 were only minor adducts. Another N7-trihydroxybutylguanine adduct (G3), which could not unambiguously be identified but is either another isomer of N7-2,3,4-trihydroxybutylguanine or, more likely, N7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent in rats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD is G4, which is formed from EBD, and not from DEB.     

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.     

Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.