Birth is characterized by an intense oxidative stress resulting in nucleotide alterations and gene overexpression in mouse lung. We showed that cigarette smoke (CS) is carcinogenic when exposure starts soon after birth and applied this bioassay to evaluate the efficacy of chemopreventive agents. The present study evaluated whether administration of the antioxidants N-acetyl-L-cysteine (NAC) and vitamin C or ascorbic acid (AsA) during pregnancy can protect strain H Swiss mice exposed to CS after birth. Exposure to CS, for 4 months, of newborns from untreated mice resulted in significant alterations at 8 months of life, including alveolar epithelial hyperplasia, emphysema, blood vessel proliferation, microadenomas, adenomas, and malignant tumors in lung, liver parenchymal degeneration and urinary bladder epithelium hyperplasia. Treatment throughout pregnancy with either NAC, a scavenger of reactive oxygen species, or AsA, an electron donor, did not affect fertility, parity, and body weight of newborns. Prenatal antioxidants significantly inhibited most lesions in adult mice exposed to CS since birth. For instance, the incidence of emphysema was reduced from 27.5% in CS-exposed mice that were untreated during pregnancy to 7.1% and 14.0% in those treated prenatally with NAC and AsA, respectively. Lung adenomas were reduced from 34.8% to 16.7% and 9.3%, respectively. Malignant lung tumors were reduced from 13.0% to 4.7% by prenatal AsA. Liver parenchymal degeneration was reduced from 58.0% to 14.3% by prenatal NAC. These data mechanistically support a "transplacental chemoprevention" strategy, aimed at protecting the newborn from oxidative stress and the adult from CS-related diseases appearing later in life.

The Fhit gene, encompassing the most active common human chromosomal fragile region, FRA3B, has been shown to act as a tumor suppressor. Several studies have shown significant Fhit alterations or Fhit protein loss in lung cancers from smokers compared with lung cancers from nonsmokers. To evaluate the role of Fhit under controlled experimental conditions, we exposed rodents to environmental cigarette smoke (ECS) and evaluated Fhit expression or Fhit protein in the respiratory tract. After 14 days of exposure to ECS, loss of Fhit protein in the bronchial/bronchiolar epithelium affected half of the tested B6-129(F(1)) mice, either wild type or Fhit(+/-). After 28 days, it affected the vast majority of the tested SKH-1 hairless mice and of A/J mice and all (UL53-3 x A/J)F(1) mice, either wild type or P53(+/-). In Sprague-Dawley rats, exposure to ECS for up to 30 days caused a time-dependent loss of Fhit in pulmonary alveolar macrophages. Moreover, ECS down-regulated Fhit expression and significantly decreased Fhit protein in the rat bronchial epithelium. The oral administration of N-acetylcysteine attenuated the ECS-related loss of Fhit, whereas oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate, and indole 3-carbinol, and their combinations had no significant effect. Parallel studies evaluated a variety of molecular, biochemical, and cytogenetic alterations in the respiratory tract of the same animals. In conclusion, there is unequivocal evidence that Fhit is an early, critical target in smoke-related lung carcinogenesis in rodents, and that certain chemopreventive agents can attenuate the occurrence of this gene alteration.

The epidemic of lung cancer and the increase of other tumours and chronic degenerative diseases associated with tobacco smoking have represented one of the most dramatic catastrophes of the 20th century. The control of this plague is one of the major challenges of preventive medicine for the next decades. The imperative goal is to refrain from smoking. However, chemoprevention by dietary and/or pharmacological agents provides a complementary strategy, which can be targeted not only to current smokers but also to former smokers and passive smokers. This article summarises the results of studies performed in our laboratories during the last 10 years, and provides new data generated in vitro, in experimental animals and in humans. We compared the ability of 63 putative chemopreventive agents to inhibit the bacterial mutagenicity of mainstream cigarette smoke. Modulation by ethanol and the mechanisms involved were also investigated both in vitro and in vivo. Several studies evaluated the effects of dietary chemopreventive agents towards smoke-related intermediate biomarkers in various cells, tissues and organs of rodents. The investigated end-points included metabolic parameters, adducts to haemoglobin, bulky adducts to nuclear DNA, oxidative DNA damage, adducts to mitochondrial DNA, apoptosis, cytogenetic damage in alveolar macrophages, bone marrow and peripheral blood erytrocytes, proliferation markers, and histopathological alterations. The agents tested in vivo included N-acetyl-l-cysteine, 1,2-dithiole-3-thione, oltipraz, phenethyl isothiocyanate, 5,6-benzoflavone, and sulindac. We started applying multigene expression analysis to chemoprevention research, and postulated that an optimal agent should not excessively alter per se the physiological background of gene expression but should be able to attenuate the alterations produced by cigarette smoke or other carcinogens. We are working to develop an animal model for the induction of lung tumours following exposure to cigarette smoke. The most encouraging results were so far obtained in models using A/J mice and Swiss albino mice. The same smoke-related biomarkers used in animal studies can conveniently be applied to human chemoprevention studies. We participated in trials evaluating the effects of N-acetyl-l-cysteine and oltipraz in smokers from Italy, The Netherlands, and the People's Republic of China. We are trying to develop a pharmacogenomic approach, e.g. based on genetic metabolic polymorphisms, aimed at predicting not only the risk of developing cancer but also the individual responsiveness to chemopreventive agents.

Multigene‐expression analysis provides a formidable tool for evaluating cellular functions, under either physiological or pathological conditions, and for assessing their modulation by exogenous agents. We investigated multigene expression in the liver and lung of rats receiving intratracheal instillations of sodium dichromate for 3 consecutive days. Nylon membrane cDNA arrays were hybridized with standardized amounts of 32 P‐labeled probes, and the results were normalized by making reference to housekeeping genes. The basal expression of 52 of 216 tested genes was 2.1–11.1 times higher in the liver than in the lung of control rats. No alteration of gene expression occurred in the liver of chromium(VI)‐treated rats, consistent with the fact that this metal species, being reduced upstream, can exert effects only locally but not at a distance from the respiratory tract. In contrast, the expression of 56 genes was increased 2.1 to 3.0 times in the lung as an early response to chromium(VI) administration. The altered genes are involved in the metabolic reduction of chromium(VI) and in a variety of interconnected functions, such as multidrug resistance and stress response, protein and DNA repair mechanisms, signal transduction pathways, apoptosis, and cell‐cycle modulation. Thus, short‐term treatment with chromium(VI) by intratracheal administration triggered a variety of defense processes in the lung. Although the use of selected genes does not provide an exhaustive picture of overall gene expression, these findings contribute to our understanding of chromium toxicology and provide a further mechanistic support to the involvement of thresholds in chromium(VI) carcinogenesis. © 2002 Wiley‐Liss, Inc.

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.

Fermented milk products might be used for cancer chemoprevention due to their putative anticarcinogenic and antitumor activities. The diet was supplemented with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B 144 (product FFM.B 144) added throughout the experiment at doses of 1.3 g and 2.5 g per rat, 5 times a week starting 3 weeks before the first carcinogen injection. This treatment significantly inhibited, by 26.2-28.6% and by 34.2%, the total intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH, 21 mg/kg, s.c., once per week for 20 weeks) in male and female BD6 rats, respectively. FFM.B144 decreased the tumor incidence and multiplicity in large bowel, caecum, and duodenum. Protective effects were better expressed in female animals, with exception of that observed in duodenum. Supplementation of diet with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B5 (product FFM.B5) inhibited DMH-induced carcinogenesis only in the large bowel, but had no significant protective effect when all intestinal tumors were taken into account. However, both freeze-dried products favorably shifted the differentiation of large bowel tumors by increasing the proportion of benign and highly differentiated malignant tumors and decreasing in parallel the number of poorly differentiated carcinomas without influencing the tumor size. A lower number of cases with visible mesenterial metastasis was also observed in FFM-treated rats. In addition, both FFM.B 144 and FFM.B5 significantly inhibited, by 26-33%, the induction in the same rats of ear-duct tumors. FFM.B144 but not FFM.B5 was also effective in inhibiting the tracheal carcinogenesis induced in Syrian golden hamsters by diethylnitrosamine (DEN, 100 mg/kg, two s.c. injections), the protective effect being better expressed in female animals. The anticarcinogenic potential of some fermented milk products might be exploited in chemoprevention of cancer in humans.

Damage to mitochondrial DNA (mtDNA) has been postulated to be associated with aging, cancer, and other chronic degenerative diseases which are the predominant causes of death in the population. We used molecular dosimetry techniques, i.e., 32P postlabeling and synchronous fluorescence spectrophotometry, in order to evaluate the formation of adducts to both mtDNA and nuclear DNA (nDNA) in different organs of rats exposed to genotoxic carcinogens. Adducts to mtDNA were detected following administration of benzo(a)pyrene i.p. or 2-acetylaminofluorene by gavage as well as following exposure of animals to cigarette smoke. mtDNA adduct levels were consistently higher than those to nDNA in the same cells, and qualitative differences were also pointed out in the case of the aromatic amine. The oral administration of the thiol N-acetylcysteine, one of the most promising cancer chemopreventive agents endowed with nucleophilic and antioxidant properties, produced a significant decrease of mtDNA adducts in the liver of 2-acetylaminofluorene-treated rats and, sharply, in the lung and liver of rats exposed to cigarette smoke.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques, In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type I I-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type I I-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

DNA adducts are biomarkers evaluating the biologically effective dose of carcinogens, which reflects, more realistically than the external exposure dose, an enhanced risk of developing cancer. Likewise, inhibition of DNA adduct formation can be assumed as an indicator of decreased risk. Molecular dosimetry techniques can be exploited in anticarcinogenicity studies in animal models as well as in Phase II clinical chemoprevention trials. We have extensively used these end points in animal studies using individual carcinogens and complex mixtures. As assessed by 32P-postlabelling, DNA adducts were formed in the liver of rats fed a diet supplemented with 2-acetylaminofluorene. DNA adducts were detected by synchronous fluorescence spectrophotometry (SFS) in rat liver, lung, heart and testis following intratracheal (l.t) instillations of benzo[a]pyrene. The whole-body exposure of rats to mainstream cigarette smoke resulted in the appearance of DNA adducts in lung, heart, aorta and kidney, whereas adducts were not detected by SFS in liver, brain, oesophagus and testis. Moreover, typical diagonal radioactive zones and multiple DNA adducts were revealed by 32P-postlabelling in the tracheal epithelium, nasal mucosa, aorta and testis of smoke-exposed rats. Formation of adducts to lung DNA, as assessed by both 32P-postlabelling and SFS, also occurred in rats receiving i.t. instillations of air particulate extracts from polluted urban and rural areas. The oral administration of the thiol N-acetylcysteine (NAC) significantly inhibited the formation of DNA adducts in all organs of the rats exposed to the aforementioned carcinogens, which correlated with the parallel inhibition of biochemical, cytogenetic and histopathological alterations as well as with inhibition of preneoplastic and neoplastic lesions in rodents. Our working hypothesis is that DNA adducts in trachea/lung, heart and aorta may be associated with lung cancer, cardiomyopathies and atherosclerosis, respectively. DNA adducts were consistently detectable in the DNA of smooth muscle cells from abdominal aorta specimens taken at surgery from atherosclerotic patients. Even broader are the consequences of mitochondrial (mt) DNA impairment, which has been associated with aging, cancer, and other degenerative diseases. Our data show that adduct levels are consistently higher in mtDNA than in the nuclear DNA in different organs of rats exposed either to benzo[a]pyrene, 2-acetylaminofluorene or cigarette smoke. NAC significantly decreased the formation of adducts to mtDNA when administered with drinking-water. Inhibition of adducts to nuclear DNA is one of the end points evaluated in ongoing Phase II chemoprevention trials in high-risk individuals.

The thiol N‐acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis‐associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type‐IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16‐F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden‐chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16‐F10 murine melanoma cells, injected i.v. into nude mice, were pre‐treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48‐72 hr before subcutaneous inoculation of either B16‐F10 cells or of their highly metastatic variant B16‐BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose‐related delay in tumor formation. Spontaneous metastasis formation by B16‐F10 and B16‐BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor‐cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol. © 1995 Wiley‐Liss, Inc.

The mutagenicities of mainstream cigarette smoke (CS), a cigarette smoke condensate (CSC) and smokers' urines were investigated by using batteries of Salmonella typhimurium and Escherichia coli strains. The S9-mediated mutagenicity of CSC was remarkably enhanced when using nitroreductase- and especially O-acetyltransferase-overproducing derivatives of the classical strains TA98 and TA100, with the following rank of sensitivity: YG1024 > YG1029 > YG1021 > TA98NR > YG1026 > TA98 > TA100 > TA100-DNP6 > TA100NR > TA98-1,8-DNP6. With YG1024, a doubling of spontaneous revertants was observed with as little as 1/110 of the smoke condensate recovered from one cigarette under our experimental conditions. Similarly, the S9-mediated mutagenicity of mainstream CS was considerably increased in YG1024 and YG1029, the O-acetyltransferase-overproducing derivatives of TA98 and TA100, respectively. In the absence of S9 mix, the concentrates of 23 urine specimens from five smokers failed to revert S. typhimurium TA98 and YG1024, and were equitoxic in E. coli WP2 and its repair-deficient counterpart CM871 (uvrA-, recA-, lexA-). In the presence of S9 mix, all specimens were mutagenic, with an average YG1024:TA98 ratio of 6.6:1. These patterns suggest that the bacterial mutagenicity of smoke-associated complex mixtures is mainly due to aromatic amines. The mutagenicities of other typical constituents of CS, i.e. the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) and the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), were not appreciably enhanced in the O-acetyltransferase-overproducing strain YG1029, compared to its parental strain TA100. Moreover, BP and NNK induced less than additive mutagenic responses when combined at high doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Due to the postulated role of mitochondrial DNA damage in aging as well as in the pathogenesis of several chronic degenerative diseases, including tumors, there is a need for easy, safe, rapid and inexpensive methods for mitochondrial DNA isolation. We propose a simple, 1-day protocol based on alkaline extraction, which avoids time-consuming gradient centrifugations and use of toxic phenol. This procedure was used for the recovery of mitochondrial DNA from different rat organs (liver, kidney, heart, lung, brain and testis). The yield was quite high. Purity was sufficient enough to perform restriction analyses with several endonucleases. No changes were observed in the structure or methylation patterns of -CCGG- sites in liver mitochondrial DNA isolated from rats exposed whole-body to mainstream cigarette smoke or treated with the carcinogens benzo[a]pyrene, 2-acetylaminofluorene or diethylnitrosamine.

The ability of the aminothiol N-acetylcysteine (NAC) to prevent the formation of carcinogen-DNA adducts in tracheal epithelial cells was investigated in Sprague-Dawley rats exposed whole-body to mainstream cigarette smoke for either 40 or 100 consecutive days. 32P-Postlabelling analyses showed the occurrence of DNA adducts (12.49 adducts/10(8) nucleotides) after 40 days of exposure, with a trend to formation of characteristic diagonal radioactive zones. Total adduct levels were not further enhanced after 100 days of exposure to smoke, although significant changes occurred in the amounts of individual adducts. NAC, given by gavage in the 40 day study and in drinking water in the 100 day study, significantly inhibited the formation of smoke-related carcinogen-DNA adducts in the tracheal epithelium, to such an extent that adduct levels were not significantly higher than those detected in sham-exposed control rats. Together with a variety of other molecular, clastogenicity, metabolic, cytological and histopathological end-points investigated in rodents and with the preliminary evidence arising from a study in humans, these results document the considerable efficacy of oral NAC in inhibiting smoke-related carcinogen-DNA adducts.

A series of naturally occurring compounds were tested for the ability to modulate the mutagenicity induced by cigarette smoke (CS), cigarette smoke condensate (CSC) and benzo[a]pyrene (BP) in the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow. Sodium selenite, retinol acetate and alpha-tocopherol significantly decreased the mutagenic activity of CS in Salmonella typhimurium TA98. Ascorbic acid, reduced glutathione (GSH), cysteine, caffeine, theophylline, cobalt chloride, folic acid, adenine, adenosine, guanosine, cytidine and cytosine were conversely devoid of any significant effect. Sodium selenite slightly decreased the mutagenic activity of CSC in the same bacterial strain, while caffeine was ineffective and ascorbic acid potentiated its mutagenicity. Ascorbic acid inhibited the mutagenic activity of BP in S. typhimurium TA98, but not in TA100. Retinol acetate diminished the number of BP-induced his+ revertants in TA98 but only at the highest concentrations used, whereas alpha-tocopherol, GSH, cysteine, sodium selenite and caffeine had no effect. Selenite and GSH, which were ineffective when applied individually, inhibited in a dose-dependent manner the BP-induced mutagenesis in S. typhimurium TA98 when simultaneously added to the top agar. All other combinations tested, including selenite plus either GSH, cysteine or caffeine towards CS or CSC, or selenite plus cysteine, or selenite plus retinol acetate and alpha-tocopherol towards BP, failed to produce interactive effects. Sodium selenite and caffeine, given either alone or in combination in drinking water, did not influence the clastogenesis induced in mouse bone marrow by a single treatment with CS or BP. Ascorbic acid was also ineffective towards CS clastogenicity but significantly decreased the number of micronucleated polychromatic erythrocytes induced by BP.(ABSTRACT TRUNCATED AT 250 WORDS)

Isorbide-5-mononitrate and isosorbide-2-mononitrate, the metabolites of isosorbide dinitrate, were studied for their effects on the contractile and electrical activity and on the content of cyclic nucleotides in isolated heart muscles of guinea-pig and dog. Isosorbide-5-mononitrate at all concentrations tested and isosorbide-2-mononitrate at low concentrations did not produce significant changes in the mechanical activity of guinea-pig heart preparations. Isosorbide-2-mononitrate in concentrations above 10(-4) M inhibited the contractility and shortened the action potential of guinea-pig and dog heart muscle. Neither mononitrate had an effect on cAMP concentration but both induced a significant elevation of cGMP content in guinea-pig heart tissue, the effect of isosorbide-2-mononitrate being greater.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.

Pre-treatment, co-treatment, and post-treatment procedures were comparatively used in order to assess the modulation of mutagenicity in S. typhimurium his- strains. Pre-treatment of bacteria with sodium selenite had no effect on sodium azide mutagenicity. Irrespective of the procedure used neither selenite nor caffeine had any influence on the S9-mediated mutagenicity of aflatoxin B1. In contrast, the mutagenicity of N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) was variably affected, depending on the sequence of exposures of target bacterial cells to mutagens and modulators. In particular, pre-treatment of bacteria with either selenite or caffeine or their combination generally resulted in a potentiation of MNU and MNNG mutagenicity. However, co-incubation of these alkylating agents and test modulators with bacterial cells yielded an evident inhibition of mutagenicity, the methylxanthine being more effective in this case. Caffeine exhibited an an antimutagenic effect towards MNU also when assayed in a post-treatment procedure. Thus, in dependence on the test conditions, selenite and caffeine could act in the same mutagenicity assay as co-mutagens, antimutagens or agents without effect on mutagenesis. These opposite trends reflect the complexity of the mechanisms of action of both mutagens and modulators tested, and underscore the variable outcome of their interactions, also depending on topological and chronological factors. The data reported emphasize the need for a multiple methodological approach in studies investigating the modulation of mutagenicity.