Genotoxicity evaluation of selenium sulfide in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays

Selenium monosulfide (Ses) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/ in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.     

Effect of pre-exposure to beta rays of tritium on some biochemical parameters measured in organs of rats subsequently irradiated with fast neutrons

The experiment examined biological responses produced by combined sequential exposure to low-level tritium contamination, followed by challenging irradiation with fast neutrons. Modifications of endogenous antioxidant potential of different organs in rats were discussed in relation to tissue radiosensitivity. Rats pre-contaminated to 7 cGy and 35 cGy have been additionally irradiated to 1 Gy with fast neutrons. Lipid peroxide level was determined in liver, kidney, small intestine, spleen, bone marrow, and plasma. Reduced glutathione (GSH) level and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in erythrocytes. An in vitro thymidine uptake assay was performed in isolated bone marrow cells. The lipid peroxide level decreased significantly only in liver and kidney from rats pre-exposed to 35 cGy. For small intestine and spleen, tissues of comparatively higher radiosensitivity, no induced radioprotection was observed, as reflected in the homeostasis of the lipid peroxides. The same behavior was observed in bone marrow, the most radiosensitive tissue studied. However, the bone marrow thymidine-incorporation assay revealed a possible adaptive-type reaction in rats pre-exposed to 35 cGy. We conclude that for radiosensitive tissues pre-exposure to chronic low doses of low linear energy transfer (LET) irradiation has no protective effect on their antioxidant status, whereas a protective effect is observed in radioresistent tissues.     

The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: the response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry

The frequency of micronucleated polychromatic erythrocytes (fMPCE) was determined in samples from bone marrow, spleen and peripheral blood of rats exposed to low doses of X-rays, cyclophosphamide or vincristine. The fMPCE values were lower in the peripheral blood than in bone marrow or spleen. This is due to the elimination of MPCE from the circulating blood, which was confirmed by the results from prolonged exposure of rats to gamma-radiation. When the analysis was restricted to the youngest PCE in peripheral blood, the sensitivity of the assay was considerably improved. This can be reproducibly achieved with the flow cytometric analysis.     

A lacZ transgenic mouse assay for the detection of mutations in follicular granulosa cells

There is ongoing concern that an assay for germ cell effects in female animals is not available. While transgenic mutation detection systems provide unprecedented access to numerous rodent tissues, studies on the induction of gene mutations in oocytes are still not possible because sufficient numbers of cells cannot be harvested. However, following stimulation of an ovarian follicle, the granulosa cells contained therein divide rapidly, increasing substantially in numbers. Since these granulosa cells share the same environment as the ovum, they may serve as suitable surrogates for the study of exposure of female germ cells to mutagens. Female lacZ transgenic mice (MutaMouse) were treated by intraperitoneal injection of N-ethylnitrosourea (ENU) and subsequently with pregnant mare serum gonadotropin (PMSG, 5IU/animal, i.p.) to induce follicular growth. Animals were sacrificed 48 h after the administration of PMSG and granulosa cells and bone marrow were harvested. A comparable dose-related increase in the mutant frequency (MF) of both granulosa and bone marrow cells was observed. The highest dose caused a decrease in the MF of granulosa cells, but not in the bone marrow, suggesting possible greater susceptibility of granulosa cells to ENU toxicity. Doubling dose estimates for bone marrow and granulosa cells were lower than those derived from the literature on oocyte mutation frequency using the Russell specific locus assay, suggesting that both cell types are more sensitive to ENU-induced mutation than oocytes. The results indicate that transgene mutations in granulosa cells may provide a sensitive pre-screening tool for potential genotoxic germ cell effects of exposed oocytes.     

Cytosolic enzymes from rat tissues that activate the cooked meat mutagen metabolite N-Hydroxyamino-1-methyl-6- phenylimidazo[4,5-b]pyridine (N-OH-PhIP)

Heterocyclic amines are formed during the cooking of foods rich in protein and can be metabolically converted into cytotoxic and mutagenic compounds. These "cooked-food mutagens" constitute a potential health hazard because DNA damage arising from dietary exposure to heterocyclic amines can modify cell genomes and thereby affect future organ function. To determine enzymes responsible for heterocyclic amine processing in mammalian tissues, we performed studies to measure genotoxic activation of the N-hydroxy form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) --a common dietary mutagen. O-Acetyltransferase, sulfotransferase, kinase, and amino-acyl synthetase activities were assayed using substrate-specific reactions and cytosolic enzymes from newborn and adult rat heart, liver, spleen, kidney, brain, lung, and skeletal muscle. The resultant enzyme-specific DNA adduct formation was quantified via (32)P-postlabeling techniques. In biochemical assays with rat tissue cytosolic proteins, O-acetyltransferases were the enzymes most responsible for N-hydroxy-PhIP (N-OH-PhIP) activation. Compared to O-acetyltransferase activation, there was significantly less kinase activity and even lesser amounts of sulfotransferase activity. Proyl-tRNA synthetase activation of N-OH-PhIP was not detected. Comparing newborn rat tissues, the highest level of O-acetyltransferase mutagen activation was observed for neonatal heart tissue with activities ranked in the order of heart > kidney > lung > liver > skeletal muscle > brain > spleen. Enzymes from cultured neonatal myocytes displayed high O-acetyltransferase activities, similar to that observed for whole newborn heart. This tissue specificity suggests that neonatal cardiac myocytes might be at greater risk for damage from dietary heterocyclic amine mutagens than some other cell types. However, cytosolic enzymes from adult rat tissues exhibited a different O-acetyltransferase activation profile, such that liver > muscle > spleen > kidney > lung > brain > heart. These results demonstrated that enzymes involved in catalyzing PhIP-DNA adduct formation varied substantially in activity between tissues and in some tissues, changed significantly during development and aging. The results further suggest that O-acetyltransferases are the primary activators of N-OH-PhIP in rat tissues.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

Abstract We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2–3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (<2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found that the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

Effect over time of in-vivo administration of the polysaccharide arabinogalactan on immune and hemopoietic cell lineages in murine spleen and bone marrow.

Current evidence indicates an immunostimulating role for complex carbohydrates, i.e., polysaccharides, from several plant sources. In the present work, we determined the specific in vivo effects, with time of administration, of one such compound, a neutral arabinogalactan from larch not only on immune (lymphoid) cells, but also on natural killer (NK) lymphoid cells, as well as a variety of other hemopoietic cells in both the bone marrow and spleen of healthy, young adult mice. The latter were injected daily (i.p.) with arabinogalactan (500 microg in 0.1 ml pH 7.2 phosphate buffered saline-PBS) for 7 or 14 days. Additional, aged (1 1/2-2 yr) mice were similarly injected for 14 days only. Control mice were given the PBS vehicle in all cases, following the above injection regimen. Animals from all groups were sampled 24 h after the final injection and the immune and hemopoietic cell populations in the bone marow and spleen were assessed quantitatively. The results indicated that immediately following either 7 or 14 days of arabinogalactan administration to young, adult mice, lymphoid cells in the bone marrow were significantly decreased (p < 0.004; p < 0.001, respectively) relative to controls but remained unchanged at both time intervals in the spleen. NK cells, after 7 days of arabinogalactan exposure, were also decreased significantly in the bone marrow (p < 0.02), but unchanged in the spleen. After 14 days' exposure to the polysaccharide, NK cells in the bone marrow had returned to normal (control) levels, but were increased in the spleen (p < 0.004) to levels greater than 2-fold that of control. Among other hemopoietic cell lineages, none was influenced in the bone marrow or spleen by one-week administration of arabinogalactan; however, after two-week exposure, precursor myeloid cells and their mature (functional) progeny (granulocytes), were significantly reduced in the spleen (p < 0.043; p < 0.006, respectively), as were splenic monocytes (p < 0.001). These lineages in the bone marrow, however, remained steadfastly unaltered even after 14 days of continuous exposure to the agent. Of the vast cascade of cytokines induced in the presence of this polysaccharide, it appears that immunopoiesis- and hemopoiesis-inhibiting ones are most prevalent during at least the first two weeks of daily exposure.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2-3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (less than 2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

Testing the Genotoxicity,Cytotoxicity, and Oxidative Stress of Cadmium and Nickel and Their Additive Effect in Male Mice

The present study was aimed to investigate the ability of cadmium (Cd) and nickel (Ni) to induce genotoxicity, cytotoxicity, and oxidative stress in bone marrow cells of male mice. Aneuploidy and chromosomal aberrations (CA) showed that Cd is a stronger mutagen than Ni. Cd and Ni increased significantly the incidences of micronucleated polychromatic erythrocytes (PCEs). Also, the ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) suggests that treatment with higher doses of the two metals increased the cytotoxicity. Numerical chromosomal aberrations increased hypoploidy with the treatment which reached two to three times of the frequency of hyperploidy. The results showed that both Cd and Ni are aneugenic that act on kinetochores and cause malsegregation of chromosomes as well as being clastogenic. Both Cd and Ni increased single-break aberrations and also Cd and Ni were found to induce significant DNA damage in mouse bone marrow cells as assessed by the comet assay. In addition to the cytotoxicity results, biochemical analysis in bone marrow revealed a dose-dependent increase of oxidative stress markers. According to the results obtained, genotoxicity and cytotoxicity effects of cadmium and nickel in vivo are dose-dependent and are associated with oxidative stress and their combined effect is less than their expected additive effect, and it could be concluded that there are no synergistic effects resulting from the combined application of both metals.     

Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues

Rio Grande do Sul (RS) coal is low quality and typically obtained by strip mining. In a recent study concerning 2 years of biomonitoring in coal regions, we demonstrated the genotoxicity of coal and related products on blood cells of native rodents, from RS, Brazil. With the goal of studying the variations in the effects of RS coal on different tissues of the same rodent, we utilized, besides the single cell gel (SCG) and micronucleus (MN) assay on blood, histological analyses and SCG assay of bone marrow, spleen, kidney, liver and lung cells, and MN assay of bone marrow and spleen cells. In addition, to identify agents that can potentially influence the results, concentrations of several heavy metals were analyzed in livers and in soil, and the total concentration of hydrocarbons in the soil was determined. Rodents exposed to coal were captured at two different sites, Butiá and Candiota, in RS. Reference animals were obtained from Pelotas, where there is no coal mining. This report provides chemical and biological data from coal regions, indicating the possible association between Zn, Ni, Pb and hydrocarbons in the induction of DNA damage (e.g. single strand-breaks and alkali-labile sites) determined by the alkaline SCG assay in cells from Ctenomys torquatus. The results of the present SCG study indicate that coal and by-products not only induce DNA damage in blood cells, but also in other tissue cells, mainly liver, kidney and lung. Neither the MN assay nor histopathological observations showed significant differences; these analyses may be useful under circumstances where genotoxicity is higher. In conclusion we believe that the in vivo genotoxicity of coal can be biomonitored by the SCG assay, and our studies suggest that wild rodents, such as C. torquatus are useful for monitoring genotoxic damage by both methods, the SCG assay and the MN test.     

Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene

Oral dosing of between 5–30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of > 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4–36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal.

It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should also be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.     

Effect of whole-body gamma irradiation on lipid peroxidation in rat tissues

In this work, we studied the influence of wholebody gamma irradiation (800 rads) upon malonaldehyde (MDA) content in plasma, erythrocyte, brain, heart, lung, kidney, spleen, liver, thymus and bone marrow. MDA levels were increased in all studied samples, except lung; the highest increases were observed in the most radiosensitive organs (bone marrow, thymus, spleen) and not in those continuously exposed to high concentrations of molecular oxygen (lungs, erythrocytes). Comparison of the variations of MDA levels in plasma, kidneys and spleen to those in the other tissues lead to the hypothesis that MDA is released from tissues in plasma and trapped from plasma in kidney and spleen. The variations in plasma and erythrocyte were found not to be related to each other.     

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.     

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Gene transfer to suppress bone marrow alkylation sensitivity

Alkylating agents represent a highly cytotoxic class of chemotherapeutic compounds that are extremely effective anti-tumor agents. Unfortunately, alkylating agents damage both malignant and non-malignant tissues. Bone marrow is especially sensitive to damage by alkylating agent chemotherapy, and is a dose-limiting tissue when treating cancer patients. One strategy to overcome bone marrow sensitivity to alkylating agent exposure involves gene transfer of the DNA repair protein O(6)-methylguanine DNA methyltransferase (O(6)MeG DNA MTase) into bone marrow cells. O(6)MeG DNA MTase is of particular interest because it functions to protect against the mutagenic, clastogenic and cytotoxic effects of many chemotherapeutic alkylating agents. By increasing the O(6)MeG DNA MTase repair capacity of bone marrow cells, it is hoped that this tissue will become alkylation resistant, thereby increasing the therapeutic window for the selective destruction of malignant tissue. In this review, the field of O(6)MeG DNA MTase gene transfer into bone marrow cells will be summarized with an emphasis placed on strategies used for suppressing the deleterious side effects of chemotherapeutic alkylating agent treatment.     

Effects of a low-pathogen environment on natural killer cells of normal and B lymphocyte-deficient mice.

The effect of short-term exposure (1 month) to a low-pathogen environment (LPE) on NK cell levels in the bone marrow and spleen of immunologically normal CBA/CaJ and B-lymphocyte-deficient CBA/N mice was assessed using both functional (51Cr release) and histological methods. The total NK activity (TNKA), i.e. the percentage specific lysis corrected for changes in whole organ cellularity, of the spleens of LPE-exposed CBA/CaJ mice was not significantly different from that of conventionally reared control mice of that strain (112%), while TNKA of the bone marrow of LPE-exposed mice fell to 27% of that of the bone marrow of conventionally reared controls. TNKA of the spleen and bone marrow of LPE-exposed CBA/N mice was reduced (30%) and elevated (120%), respectively, relative to their conventionally reared counterparts. The numbers of asialo-GM-1-positive (ASGM-1+) lymphoid cells, putative NK cells, in CBA/CaJ spleens were more numerous in conventionally reared than in LPE-exposed mice (4.9 X 10(6) vs. 2.7 X 10(6), and were also more numerous in the bone marrow of conventionally reared mice than in LPE-exposed animals (8.0 X 10(4), vs. 3.0 X 10(4) cells). Similarly, in LPE-exposed CBA/N mice, the numbers of ASGM-1+ lymphoid cells in the spleens and bone marrow were lower (2.7 X 10(6) and 5.4 X 10(4), respectively) than those of the spleens and bone marrow of their conventionally reared counterparts (3.8 X 10(6) and 10.0 X 10(4), respectively). The results demonstrate that short-term maintenance in an LPE affected the NK cells of both the spleen and bone marrow of immunologically normal and B-lymphocyte-deficient mice in a strain-specific manner and suggest that the external environment may regulate NK cell production and turnover.     

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.     

Relationship between large and small tumor-binding cells in the spleen and bone marrow.

By employing a distinctive feature of natural killer (NK) cells, i.e., spontaneous target cell binding, the present study aimed to follow a relatively large cohort of target-binding cells (TBC), subdivided according to size and the presence of the radioautographically labelled nucleotide, tritiated thymidine, incorporated during a 1- or 6-hour exposure period. Labelling among all TBC in the spleen was 5% by 1 h after a single injection of the isotope and this value did not change significantly after 6 h of isotope exposure. There was an insignificant increase in the labelling index of spleen-localized, labelled, large TBC throughout and beyond the isotope exposure period for at least 48 h, concomitant with a significant increase in the labelling index of spleen-localized, small TBC during the same period. In the bone marrow, small TBC showed only 2% labelling by 1 h after a single injection of isotope, while 21% of large TBC in that organ were synthesizing DNA during the same period. The results are consistent with a precursor-product relationship between large and small TBC within the bone marrow but not the spleen, and with a bone marrow-to-spleen migration of small (+/- large) TBC. Moreover, a minor population of large TBC was detected in the spleen with kinetic characteristics distinct from those of the bone marrow.