DNA damage and mutagenesis induced by procarbazine in lambda lacZ transgenic mice: evidence that bone marrow mutations do not arise primarily through miscoding by O6-methylguanine

The DNA damaging and mutagenic activities of procarbazine, a methylating drug employed in cancer chemotherapy and suspected of causing therapy-related leukaemia, were investigated in the liver and bone marrow of lambda lacZ transgenic mice (MutaMouse). The drug was administered using two different protocols, a 'high-dose' one involving 5 daily doses of 200 mg/kg, expected to cause depletion of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) and thus favour the selective accumulation of the premutagenic lesion O6-methylguanine (O6- meG) relative to other adducts, and a 'low-dose' one involving 10 daily doses of 20 mg/kg procarbazine. Substantial accumulation of O6-meG was observed in both tissues examined 6 h after the end of the 'high-dose' treatment, with the liver accumulating somewhat higher levels than the bone marrow (28.0 +/- 1.8 fmol/microg DNA and 18.5 +/- 1.1 fmol/microg DNA respectively). However, significant increases in mutant frequency 10 days after the end of treatment were observed only in the bone marrow, reaching a 16-fold increase over background following the 5 x 200 mg/kg treatment. Sequence analysis of the mutations induced after this treatment revealed a mixed spectrum, in which G:C-->A:T transitions (characteristic of O6-meG miscoding) were only a secondary feature: Among 20 mutants analysed, only six such mutations were found, including three at CpG sites, which might have arisen from deamination of 5-methylcytosine. The other mutations observed included 1 A:T-->G:C transition, five transversions (one G:C-->T:A, one double G:C-->C:G, two A:T-->T:A, one A:T-->C:G), five deletions and three insertions. The mechanistic and clinical significance of these findings is discussed.      

DNA adducts, mutant frequencies and mutation spectra in lambda lacZ transgenic mice treated with N-nitrosodimethylamine

Groups of lambda lacZ transgenic mice were treated i.p. with N- nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in various tissues. Adduct induction in the liver exceeded by at least one order of magnitude than observed in the next nearest target tissue (lung), and was approximately linearly related to dose, except for O6-methylguanine after the first dose of 1 mg/kg which was lower than expected. Substantial induction of lambda lacZ mutagenesis was observed only in the liver, where the mutant frequency was already maximal within 7 days after 5 mg/kg NDMA and remained unchanged thereafter up to 49 days. Small but marginally significant increases in mutant frequency were consistently observed in the spleen after all three modes of treatment. A lack of proportionality between mutation induction and the administered dose or the corresponding adduct levels was observed, probably reflecting the importance of toxicity-related cell proliferation caused by NDMA at higher doses. Twenty eight days after a dose of 10 mg/kg (causing a 3.6- fold increase in mutant frequency), NDMA was found to increase the frequency of GC-->AT mutations (with a concomitant shift of their preferential location from CpG sites to GpG sites), which made up approximately 60% of the induced mutations. Surprisingly, NDMA also caused a significant increase in deletions of a few (up to 11) base- pairs (22%).      

In vivo and in vitro genotoxicity of several N-nitrosamines in extrahepatic tissues of the rat.

Toxicological mechanisms involved in organotropism of tumor induction may include cell-specific metabolic activation of the carcinogen, in vivo distribution of active metabolites and persistance of induced DNA damage. In order to elucidate which factors are involved in the organotropic action of environmentally relevant N-nitrosamines, we have studied their genotoxic and cytotoxic effects within primary intact cells of lung and kidney. The end-points determined were cytotoxicity by trypan blue exclusion and DNA single-strand break (SSB) induction by alkaline filter elution. The assays were performed in vitro to determine organ-specific metabolic activation by incubating the cells with the test compounds. The results obtained with N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDEIA), N-nitrosoethylvinylamine (NEVA), N-nitrosodibutylamine (NDBA), N-nitrosobutylbutanolamine (NBBOH), N-nitrosobutylcarboxypropylamine (BCPN), N-nitrosomethylbenzylamine (NMBzA) and N-nitrosodibenzylamine (NDBzA) indicate that several compounds may be activated to reactive metabolites by cells of kidney and lung, NDBzA revealing the highest degree of cytotoxicity. In contrast, genotoxicity in kidney cells was induced only by NBBOH and BCPN and at relative low levels. Primary lung cells could not be employed as indicators for genotoxic effects in vitro because the cell yield was not sufficient to perform the alkaline elution assay. To assess the distribution of NDMA in the whole rat and the persistence of the induced DNA damage in the two organs, further studies were carried out after oral application of 1, 2, 4, 10, 20, 32 and 40 mg/kg to the animals. Following 1 h exposure of rats to NDMA, the lowest effective genotoxic dose for lung and kidney was 2 mg NDMA/kg body wt. A plateau was achieved after a dose of 20 mg/kg in both organs. Furthermore, the persistence of DNA damage was studied in the lung. After 4 h exposure, DNA damage was still detectable at 32 mg NDMA/kg, but for the lower doses it was reduced nearly to control levels. After 16 h exposure the SSB rate in lung cells was reduced for all dose levels except for the highest dose of 40 mg/kg.     

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats [published erratum appears in Carcinogenesis 1998 Apr;19(4):707]

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.      

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase eta is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase eta in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase eta-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase eta expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase eta at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase eta. These results indicate that polymerase eta is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase eta, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.     

Molecular analysis of lacI mutants from transgenic fibroblasts exposed to 1,2-epoxybutene

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites: 1,2-epoxybutene (EB) and 1,2:3,4- diepoxybutane (DEB). We reported previously that lacI transgenic mice exposed to BD had an increased frequency of specific base substitution mutations in the bone marrow and spleen relative to unexposed controls. In the experiments described here, we determined the mutagenicity and mutational spectrum of EB in Rat2 lacI transgenic fibroblasts as a means of assessing the contribution of this metabolite to the lacI mutational spectrum of BD. Rat2 cells were exposed to 0, 0.4, 0.6, 0.8 or 1.0 mM EB for 24 h, resulting in a range of cell survival from 100 to 15%, respectively. Mutagenicity was assessed at 0, 0.6 and 1.0 mM EB. Unexposed controls had a background mutant frequency of 6 +/- 1 +/- 10(-5), while the mutant frequency in cells exposed to 0.6 and 1.0 mM EB was increased 2- and 3-fold, respectively. DNA sequence analysis of 154 lacI mutants recovered in these experiments revealed an increase in the frequency of specific base substitution mutations in cells exposed to 1.0 mM EB compared with controls. These included G:C-->A:T transitions at non-CpG sites, G:C-->T:A transversions and A:T-->T:A transversions, which have all been observed in lacI mutants isolated from transgenic mice exposed to BD. These results suggest that EB causes mutation primarily by base substitution and that the spectrum of these mutations closely resembles that of BD. These data, along with previous findings from our laboratory, suggest that EB is more likely than DEB to be primarily responsible for the lacI mutational spectrum observed in lacI transgenic mice exposed to BD.      

Organ specificity of the bladder carcinogen 4-aminobiphenyl in inducing DNA damage and mutation in mice

Aromatic amines are a widespread class of environmental contaminants present in various occupational settings and tobacco smoke. Exposure to aromatic amines is a major risk factor for bladder cancer development. The etiologic involvement of aromatic amines in the genesis of bladder cancer is attributable to their ability to form DNA adducts, which upon eluding repair and causing mispairing during replication, may initiate mutagenesis. We have investigated the induction of DNA adducts in relation to mutagenesis in bladder and various nontarget organs of transgenic Big Blue mice treated weekly (i.p.) with a representative aromatic amine compound, 4-aminobiphenyl (4-ABP), for six weeks, followed by a six-week recovery period. We show an organ-specificity of 4-ABP in inducing repair-resistant DNA adducts in bladder, kidney, and liver of carcinogen-treated animals, which accords with the bioactivation pathway of this chemical in the respective organs. In confirmation, we show a predominant and sustained mutagenic effect of 4-ABP in bladder, and much weaker but significant mutagenicity of 4-ABP in the kidney and liver of carcinogen-treated mice, as reflected by the elevation of background cII mutant frequency in the respective organs. The spectrum of mutations produced in bladder of 4-ABP-treated mice matches the known mutagenic properties of 4-ABP-DNA adducts, as verified by the preponderance of induced mutations occurring at G:C base pairs (82.9%), with the vast majority being G:C→T:A transversions (47.1%). Our data support a possible etiologic role of 4-ABP in bladder carcinogenesis and provide a mechanistic view on how DNA adduct-driven mutagenesis, specifically targeted to bladder urothelium, may account for organ-specific tumorigenicity of this chemical.     

Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells

Acrolein is an endogenous metabolite and a ubiquitous environmental pollutant. Recently, it has been suggested that acrolein is a major etiologic agent for tobacco smoking-related lung cancer. Despite the known DNA-damaging effects of acrolein, its mutagenicity to mammalian cells remains uncertain. We have investigated acrolein-induced DNA damage in relation to mutagenesis, with special focus on DNA repair, in mouse and human cells. We mapped the formation of acrolein-induced DNA adducts and the kinetics of repair of the induced lesions in the cII transgene, the mutational target, in acrolein-treated transgenic mouse fibroblasts. Acrolein-DNA adducts were formed preferentially at specific nucleotide positions, mainly at G:C base pairs, along the cII transgene. The induced acrolein-DNA adducts were moderately resistant to DNA repair. Quantification of cII mutant frequency in acrolein-treated cells, however, revealed that acrolein was not mutagenic to these cells at doses sufficient to produce DNA adducts. Determination of supF mutant frequency in DNA repair-proficient and DNA repair-deficient human fibroblasts transfected with acrolein-treated plasmids confirmed a lack of acrolein mutagenicity. Because CpG methylation may intensify acrolein-DNA adduction, we examined whether the extent of CpG methylation in the supF gene can determine acrolein-induced mutagenesis in human cells. Enhancement of acrolein-DNA adduction by methylating CpGs in the supF sequence did not elicit a mutagenic response in human fibroblasts, however. We conclude that acrolein is not mutagenic to mouse and human fibroblasts, regardless of DNA repair capacity or methylation status of CpGs, possibly because of a highly accurate replication bypass of the induced lesions.     

DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene

Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue((R)) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18. 3 +/- 4 x 10(-6) p.f.u.). (32)P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.     

CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane

The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane)has previously been shown to protect rats against 9, 10-dimethyl-1,2-benz[a]anthracene tumori-genesis, thought to be due, at leastin part, to induction of phase II detoxification. We investigatedthe ability of sulforaphane to also inhibit the phase I enzymecytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsiblefor activation of several carcinogens, including dialkylnitros-amines.Using the p-nitrophenol hydroxylation assay in microsomes fromlivers of acetone-treated Sprague-Dawley rats, sulforaphanewas shown to be a potent competitive inhibitor of CYP2E1 witha K₁pg of 347.0 ± 4.5 µM. In view of this result,we studied the capacity of sulforaphane to inhibit the genotoxicityof N-nitrosodimethylamine (NDMA). Sulforaphane at concentrationsof >0.8 µM inhibited the mutagenicity of NDMA (4.4mg/plate) in Salmonella typhimurium strainDTA100 after pre-incubationfor 45 min with cytosol extract from livers of Balb/c mice pre-treatedwith acetone. Unsecheduled DNA synthesis induced by NDMA (33.5µM)in mouse hepatocytes was inhibited in a dose-dependentmanner by sulforaphane at 0.064–20 µM. Sulforaphanewas unable to inhibit mutagenicity of sodium azide (5 µg/plate),a direct acting mutagen, in the Salmonella assay. It was notitself genotoxic in hepatocytes, as measured by unscheduledDNA synthesis, or mutagenic in the strain of Salmonella employedand cytotoxic only at high concentrations (     

二甲基亚硝胺诱导大鼠DNA O^6—甲基鸟嘌呤的免疫组化研究

本文应用双PAP法对二甲基亚硝胺(N-nitrosodjmethylamine,NDMA)诱导大鼠DNAO~6—甲基鸟嘌呤(O~5—methylguanine,O~6—mG)进行了研究。分别给大鼠一次腹腔注射NDMA 1,2,3,5,10,30,50mg/kg,5小时后大鼠产生O~6—mG具有明显的剂量反应关系以及器官和细胞特异性。随着NDMA剂量的增加,产生O~6—mG的器官种类和细胞类型增加,阳性细胞形成O~6—mG的量也增加。小剂量(1—5mg/kg)NDMA仅见肝组织的细胞形成O~6—mG,NDMA剂量达50mg/kg时,肝、肾、肺、气管,食管和鼻咽组织的细胞均检出O~6—mG。     

Regulation of renal N-nitrosodimethylamine demethylase activity by androgens, progestin, glucocorticoid, and estrogen in BALB/c mice

Studies from this laboratory have shown that renal N-nitrosodimethylamine demethylase (NDMA demethylase), an enzyme responsible for the metabolism of NDMA, shows age- and sex-related differences and is modulated by testosterone. These results have been positively correlated with NDMA-induced mutagenicity and carcinogenicity studies; however, relatively large doses of testosterone (100 mg/kg or 2 mg/day) had been used. The objective of this study was to determine whether this tissue specific increase in renal NDMA demethylase was evident only with pharmacological doses or could be observed at lower doses of testosterone, and, to determine whether this testosterone-induced increase in renal NDMA demethylase was specific for androgenic hormones. BALB/c mice were given injections s.c. of varying doses of testosterone (0.01 to 1.5 mg/day) in 0.1 ml of peanut oil for 1, 4, 8, or 16 days; control mice received peanut oil alone. A 3-fold increase in renal NDMA demethylase was observed within 24 h with 0.01 mg of testosterone. Dose-dependent increases of 3-, 5-, and 11-fold were observed at 0.01, 0.05, and 0.10 mg of testosterone, respectively. Hepatic NDMA demethylase was not affected. To determine whether this induction was specific for androgenic hormones, several hormones (androgens, estrogen, glucocorticoid, and progestin) were used. A 5-, 7-, and 2-fold stimulation of renal NDMA demethylase activity was observed in female mice with 5 alpha-dihydrotestosterone, synthetic androgen methyltrienolone, and androgen agonist medroxyprogesterone acetate, respectively. The biologically inactive 5 beta-dihydrotestosterone, stereomer of 5 alpha-dihydrotestosterone, had no effect. Medroxyprogesterone, methyltrienolone, testosterone, and 5 alpha-dihydrotestosterone exert their effect by interacting with androgen receptors; 5 beta-dihydrotestosterone does not bind to androgen receptors. Dexamethasone or estradiol treatment resulted in a significant inhibition of renal enzyme activity in male mice. None of the treatments affected hepatic NDMA demethylase activities in male or female mice. This rapid and tissue specific stimulation of renal NDMA demethylase with low doses of testosterone or with other androgen agonists seems to be androgen receptor mediated and indicates a physiological relevance of this phenomenon.     

Quantification of DNA and hemoglobin adducts of 3,4-epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol

1,3-Butadiene (BD) is a confirmed rodent carcinogen and a suspect human carcinogen that forms mutagenic epoxide metabolites during biotransformation. Species differences in the roles of individual DNA reactive intermediates in BD mutagenicity and carcinogenicity are not completely understood. Evidence suggests that 1,2:3,4-diepoxybutane (DEB) is responsible for the mutagenic effect induced by exposures to low concentrations of BD in mice and that metabolites of 3-butene-1,2-diol (BD-diol) are involved in the mutagenicity at high exposures in both mice and rats. Two reactive metabolites, 3,4-epoxy-1,2-butanediol (EB-diol) and hydroxymethylvinyl ketone (HMVK), are formed during the biotransformation of BD-diol and could potentially be involved in BD-diol associated mutagenicity. To examine the role of EB-diol in BD-diol mutagenicity we have evaluated the dosimetry of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) and N-(2,3,4-trihydroxybutyl)valine (THB-Val) in female B6C3F1 mice and female F344 rats exposed by inhalation to 0, 6, 18 and 36 p.p.m. BD-diol for 4 weeks (6 h/day x 5 days/week). Results showed higher levels of both THB-Gua and THB-Val in mice than in rats. An evaluation of THB-Gua adducts showed virtually no differences between liver and lung for either species, suggesting that EB-diol is stable and is freely circulated. The data also indicated that THB adduct formation began to plateau around 18 p.p.m. in both species. Most importantly, the shape of the dose-response curve for THB adduct formation mimicked the one observed for hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutation frequency. This showed that THB adducts, which are not thought to be responsible for causing the mutations, are good quantitative indicators of mutagenicity in rodents exposed to BD-diol. Although the potential contribution of HMVK still needs to be evaluated, the data suggest that EB-diol is responsible, at least in part, for BD-diol associated mutagenicity in rodents.     

Mutation and formation of methyl- and hydroxylguanine adducts in DNA caused by N-nitrosodimethylamine and N-nitrosodiethylamine with UVA irradiation

Previously we reported that when Escherichia coli was treated with N- nitrosodialkylamine under irradiation with near UV light, mutagenesis of the bacteria took place: there was no requirement for metabolic activation. We have now studied the spectra of mutations caused by N- nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) with UVA (320-400 nm) irradiation, using standard tester strains for identifying types of mutations. Induced mutations by NDMA + UVA were the transition GC-->AT and transversions GC-->CG, GC-->TA and AT-->TA. NDEA + UVA induced mainly the GC-->CG transversion. In both cases no frameshift mutations were observed. When O6-alkylguanine-DNA alkyltransferase- deficient strains of E. coli and Salmonella typhimurium were used, the mutation levels with both NDMA + UVA and NDEA + UVA became remarkably higher than those observed with the proficient strains. We measured the O6-methylguanine (O6-meG) level in calf thymus DNA treated with NDMA + UVA. The O6-meG level was increased as a function of NDMA concentration and irradiation time. We also detected N7-methylguanine in DNA treated with NDMA + UVA. In our previous work we found formation of 8- oxodeoxyguanosine (8-oxodG) in DNA treated with N-nitrosomorpholine + UVA. The 8-oxodG/dG ratio in DNA treated with NDMA + UVA increased up to 42-fold over that of the untreated control and that in DNA treated with NDEA + UVA increased up to 67-fold. 8-OxodG formation was not affected by replacing H2O in the reaction mixture with D2O, suggesting that singlet oxygen is not the rate limiting factor in this photoactivation. We conclude that both alkylation and oxidation are involved in mutations induced by NDMA + UVA and NDEA + UVA.      

Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fusedpolycyclic aromatic hydrocarbon. CPP is highly genotoxic inbacterial and mammalian systems inducing gene mutations, sisterchromatid exchanges and morphological transformation. CPP isa mouse skin carcinogen, a mouse skin tumor initiator and inducespulmonary tumors in newborn mice. We have examined the tumorigenicactivity of CPP in strain A/J mice, have determined the formationand persistence of CPP-induced DNA adducts in lung tissue, andanalyzed the mutational spectrum in the Ki-ras oncogene fromCPP induced tumors. CPP dissolved in tricaprylin was administeredby i.p. injection to male A/J mice (20 mice/dose) at 0,10,50,100and 200 mg/kg. Animals were killed 8 months later and the lungsremoved, fixed, and surface adenomas enumerated. CPP provedto be highly tumorigenic in A/J mice in terms of inducing lungadenomas. The observed tumor multiplicities (lung adenomas/mouse)were: 97.7 ± 28.7 at 200 mg/kg, 32.8 ± 15.4 at100 mg/kg, 4.63 ± 2.11 at 50 mg/kg and 0.58 ±0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60± 0.58 lung adenomas/mouse. Groups of mice treated underthe same dosing conditions as those in the tumor studies werekilled 1, 3, 7, 14 and 21 days after treatment. The lungs wereremoved, and the DNA was subjected to DNA adduct analysis bythe 32P-postlabeling method. Total CPP-DNA adducts in mouselung peaked at day 3 with 5870 amol CPP adducts/µg DNAafter a single dose of 200 mg/kg. DNA adduct levels decreasedto 1800 amol CPP adducts/µg DNA at day 21. QualitativeDNA adduct analysis revealed four major adducts and one minoradduct. Co-chromatography of the lung DNA from CPP-treated micewith calf thymus DNA treated with CPP-3,4-oxide indicated thatall DNA adducts were oxide derived and comparison with CPP-3,4-oxlde-treatedpolydeoxyguanylic acid suggests that almost all of these adductsare CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12mutation analysis of the DNA from twnors taken from the 100and 200 mg/kg CPP dose groups demonstrated the following patterns:GGT     

Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice

The tissue distribution and macromolecular binding of benzene was studied over a dose range spanning nine-orders of magnitude to determine the nature of the dose-response and to establish benzene's internal dosimetry at doses encompassing human environmental exposures. [14C]-Benzene was administered to B6C3F1 male mice at doses ranging between 700 pg/kg and 500 mg/kg body wt. Tissues, DNA and protein were analyzed for [14C]-benzene content between 0 and 48 h post-exposure (625 Ng/kg and 5 microg/kg dose) by accelerator mass spectrometry (AMS). [14C]-Benzene levels were highest in the liver and peaked within 0.5 h of exposure. Liver DNA adduct levels peaked at 0.5 h, in contrast to bone marrow DNA adduct levels, which peaked at 12-24 h. Dose- response assessments at 1 h showed that adducts and tissue available doses increased linearly with administered dose up to doses of 16 mg/kg body wt. Tissue available doses and liver protein adducts plateau above the 16 mg/kg dose. Furthermore, a larger percentage of the available dose in bone marrow bound to DNA relative to liver. Protein adduct levels were 9- to 43-fold greater than DNA adduct levels. These data show that benzene is bioavailable at human-relevant doses and that DNA and protein adduct formation is linear with dose over a dose range spanning eight orders of magnitude. Finally, these data show that the dose of bioactive metabolites is greater to the bone marrow than the liver and suggests that protein adducts may contribute to benzene's hematoxicity.      

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.