Comparative cytogenetic analysis of bone marrow damage induced in male B6C3F1 mice by multiple exposures to gaseous 1,3-butadiene

Groups of male B6C3F1 mice (N = 12) were exposed to ambient air or to gaseous 1,3-butadiene (BD) at 6.25, 62.5, and 625 ppm for 10 exposure days (6 hr + T90/day). Exposure to BD induced in bone marrow: 1) a significant increase in the frequency of chromosomal aberrations (CA); 2) a significant elevation in the frequency of sister chromatid exchanges (SCE); 3) a significant lengthening of the average generation time (AGT); 4) a significant depression in the mitotic index (MI); and, as measured in the peripheral blood, 5) a significant increase in the proportion of circulating polychromatic erythrocytes (%PCE), and 6) a significant increase in the level of micronucleated PCE (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE). The most sensitive indicator of genotoxic damage was the frequency of SCE (significant at 6.25 ppm), followed by MN-PCE levels (significant at 62.5 ppm), and then by CA and MN-NCE frequencies (significant at 625 ppm). The most sensitive measure of cytotoxic damage was AGT (significant at 62.5 ppm), followed by %PCE (significant at 625 ppm), and then by MI (significant by trend test only). Because each cytogenetic endpoint was evaluated in every animal, a correlation analysis was conducted to evaluate the degree of concordance among the various indicators of genotoxic and cytotoxic damage. The extent of concordance ranged from a very good correlation between the induction of MN-PCE and the induction of SCE (correlation coefficient r = 0.9562) to the lack of a significant correlation between the depression in the MI and any other endpoint (r less than 0.37).     

Benzene-induced micronuclei in erythrocytes: an inhalation concentration-response study in B6C3F1 mice

High concentrations (300–1000 p.p.m) of benzene have beenshown to induce an increase in the frequency of micronucleatederythrocytes in mice. This study investigated the mutagenicityof benzene at lower concentrations, including the current limitfor occupational exposure, 1 p.p.m. The frequencies of micronucleartedpolychromatic erythrocytes (MPCE) in the bone marrow and bloodand micronucleated normochromatic erythrocytes (MNCE) in theblood of male B6C3F1 mice were Measured following inhalationof benzene at 0, 1, 10, 100 or 200 p.p.m. during an 8 week exposureperiod. Only 100 and 200 p.p.m. benzene induced a statisticallysignificant increased frequency of micronucleated erythrocytesin the bone marrow and blood. The frequency of MPCE plateauedat week 2 with 43/1000 (100 p.p.m.) and 86/1000 (200 p.p.m.)in the bone marrow as compared with 10/1000 for controls. Thefrequency of MNCE in the blood progressively increased to 13.4/1000(100 p.p.m.) and 32.5/1000 (200 p.p.m.) at week 8 as comparedwith 1.8/1000 for controls. Cytotoxicity of replicating andmaturing erythrocytes by 100 and 200 p.p.m. benzene delayedthe accumulation of MNCE in the blood. There was not a stastisticallysignificant increase in the frequency of micronucleated erythrocytes,as an indicator of mutagenicity, with inhalation of 1 or 10p.p.m. benzene over an 8 week period. A quadratic curve fitthe bone marrow MPCE data of mice exposed to up to 200 p.p.m.benzene with a high correlation (R² = 0.94) and could not berejected based on lack of fit. ¹To whom correspondence should be addressed:     

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene

1,3-Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixtures of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene. Environ. Mol. Mutagen. 29: 335–345, 1997. © 1997 Wiley-Liss, Inc.     

In vivo sister chromatid exchange and micronucleus induction studies with 1,3-butadiene in B6C3F1 mice and Sprague-Dawley rats

Male B6C3F1 mice and Sprague-Dawley rats were exposed for 2 days, 6 h/day to 1,3-butadiene (BD) by inhalation (nose only) and their bone marrow cells were evaluated for the induction of micronuclei (MN) and sister chromatid exchanges (SCEs). A significant dose-dependent increase in MN induction was observed in mice. At 100 p.p.m., the frequency of micronucleated polychromatic erythrocytes was 6-fold above control with a maximal induction of 38-fold at 10,000 p.p.m. A significant increase in SCEs was also observed in mouse bone marrow cells starting at 100 p.p.m. with a 4-fold increase over the control evident at 10,000 p.p.m. The highest tested no observed effect level for both endpoints was 50 p.p.m. In contrast, rat bone marrow cells did not exhibit significant increases in micronucleated polychromatic erythrocytes or SCEs. These results indicate that BD is genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to BD carcinogenicity.     

Micronuclei formation in somatic cells of mice exposed to 50-Hz electric fields

Male Swiss mice are tested under uniform 50-Hz electric field intensities of 100, 170, 220, and 290 kV/m. These values on the basis of equal induced current density are equivalent to the case of a human exposed to field intensities of nearly 8, 14, 18, and 24 kV/m, respectively. The latter values may be found beneath or in the vicinity of extra-high-voltage power lines whose voltages range from 220 to 765 kV. The cytogenetic effect of extremely low-frequency (ELF) electric fields, as expressed by micronuclei formation, is assessed. Mice are exposed for 24 hr, and samples are taken 48, 72, and 96 hr from the beginning of exposure. Sham-exposed mice served as controls. The number of micronucleated polychromatic erythrocytes (PCE) in exposed animals are significantly higher than those of the control. The increase in micronucleated PCE was significantly dose dependent at all times. Samples taken 96 hr after exposure show a decrease in percentages of micronucleated PCE, which may be taken as an indication of recovery.     

Sister chromatid exchange and chromosome aberrations in mice after in vivo exposure of green S--a food colorant.

Sister chromatic exchanges (SCE) and chromosome aberrations (CA) in mice after in vivo exposure of Green S were carried out following single acute treatment. Except for the lowest dose (25 mg/kg body weight) a significant increase in the SCEs were observed in all the other doses (50, 100, and 200 mg/kg) tested. In CA study two higher doses (200 and 400 mg/kg) showed a significant increase in CA when compared with control. The minimum effective dose which induced SCE and CA was 50 and 200 mg/kg of body weight, respectively. The trend tests for the evidence of dose response effects were also significant for both SCE and CA. No significant differences were observed in cell replication kinetic (RI) analysis. A significant increase in the mitotic index (MI) was also observed in the highest dose (400 mg/kg) tested when compared with control. Thus the present study indicates that Green S can induce both SCE and CA in vivo in bone marrow cells of mice.     

In vivo mutagenicity and mutation spectrum in the bone marrow and testes of B6C3F1 lacI transgenic mice following inhalation exposure to ethylene oxide

The lacI mutant frequency and mutation spectrum were determined in the bone marrow and testes of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of male transgenic lacI B6C3F1 mice were exposed to 0, 25, 50, 100 or 200 p.p.m. EO for up to 48 weeks (6 h/day, 5 days/week) and were killed at 12, 24 or 48 weeks of EO exposure for determination of lacI mutant frequency. In the bone marrow, the lacI mutant frequency was significantly increased at the two highest exposure levels (100 and 200 p.p.m.) and at the 48 week exposure time point. The shape of the exposure-response curve for lacI mutant frequency in the bone marrow was non-linear. DNA sequence analysis of the bone marrow mutation spectrum revealed that only AT-->TA transversions occurred at an increased frequency in EO-exposed mice: 25.4% in EO-exposed mice for 48 weeks (200 p.p.m.) compared with 1.4% in air controls. In testes, the lacI mutant frequency was increased at a single exposure level of 200 p.p.m. for 24 weeks. At 48 weeks, the lacI mutant frequency in testes was significantly increased to an equal degree at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. Analysis of the testes mutation spectrum in air control mice and in mice exposed to 200 p.p.m. EO for 48 weeks revealed that no single mutational type occurred at an increased frequency. In the testes, there was a small increase across all mutational types that was sufficient to increase the overall lacI mutation frequency although not significant individually. The mutation spectrum in testes of EO-exposed mice also revealed that the increased lacI mutant frequency observed at 25 or 50 p.p.m. EO was not due to an increase in mutant siblings (clonality). These data demonstrate that inhalation exposure to EO for up to 48 weeks produces distinct mutagenic responses in bone marrow and testes.     

Induction and time-dependent accumulation of micronuclei in peripheral blood of transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation

In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.     

Induction of sister chromatid exchange in multiple murine tissues in vivo by various methylating agents

In order to study the reliability of in vivo sister chromatid exchange (SCE) assays for predicting carcinogenicity, several known animal carcinogens were tested in a multicellular in vivo SCE assay and an in vivo/in vitro murine lymphocyte assay. The methylating agents 1,2-dimethylhydrazine.2 HCl (DMH), dimethylnitrosamine (DMN), methylnitrosourea (MNU), methyl methane-sulphonate (MMS), and methylazoxymethanol acetate (MAM) were tested for SCE induction in several murine tissues in vivo, including bone marrow, alveolar macrophages, regenerating and intact liver, and kidney from B6D2F1 mice. In all cell types, clear dose-responses were observed following exposure of mice to subcytotoxic fractions of the LD50 dose of DMH, MNU, or MMS. DMN (0.03-0.27 mmol/kg) produced small, although not dose-related, increases in SCE in all cell types. At the doses tested (0.06 and 0.08 mmol/kg), MAM did not induce elevated SCE in the various cell types. Following a series of multiple i.p. injections of low, non-toxic doses of DMH (0.15 mmol/kg, once a week, for 10 weeks), significant differences were observed in intact vs. regenerating liver and in single vs. multiple injections in regenerating liver. Following exposure of B6D2F1 mice to a single i.p. injection of 0.25 mmol/kg DMN, DMH, or MMS or 0.19 mmol/kg MNU, SCE responses were evaluated in Concanavalin A (Con A)- and LPS-stimulated blood and spleen lymphocytes. Considerable cytotoxicity was observed in blood lymphocytes. In Con A- and LPS-stimulated spleen lymphocytes, DMH-, and DMN- and MMS-induced SCE frequencies were approximately 1.5-2 x baseline levels and MNU-induced SCE were approximately three- to fourfold higher than baseline values in cultures initiated at 1 and 24 h postexposure. At 48 and 72 h after an i.p. injection of 0.131 mmol/kg MNU, SCE responses in lymphocytes were approximately 2 x baseline levels. At 24 h following one, two, or four injections (one/week) of 0.075 mmol/kg MNU dose-related increases in SCE were observed in spleen lymphocytes. These studies illustrate that carefully designed in vivo SCE assays may have the capacity to predict the tumorigenic potential of chemical agents.     

Effect of treatment protocol and sample time on the frequencies of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood

Studies were conducted to evaluate the effect of experimental protocol on the ability of benzidine (BZD), dimethylbenzanthracene (DMBA) and mitomycin C (MMC), administered by intraperitoneal injection, to induce micronuclei in polychromatic erythrocytes (PCE) of B6C3F1 mice. Three different treatment/sampling protocols were used, involving from one to three consecutive daily treatments and from three to one, respectively, consecutive daily samplings beginning 24 h after the last injection. DMBA and MMC elicited a significant micronucleus response in all three experimental protocols, while BZD induced a significant response only in the multiple injection protocols. Of the three protocols, the 3-day injection/single sample time protocol offers the greatest efficiency in minimizing the number of animals required in a study, in decreasing the time needed for scoring and in simplifying the statistical analysis. In addition, a comparison of the frequency of micronucleated PCE in peripheral blood and bone marrow following the treatment of mice with either BZD or DMBA suggests that, following a three injection protocol, either tissue can be used with equal efficacy.     

Evaluation of the mutagenicity of the anti-inflammatory drug salicylazosulfapyridine (SASP)

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.     

Combined treatment with 4-(N-methyl-N-nitrosamino)-1- (3-pyridyl)-1-butanone and dibutyl phthalate enhances ozone-induced genotoxicity in B6C3F1 mice

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibutyl phthalate (DBP) with ozone were investigated. Male and female B6C3F1 mice were exposed to ozone (0.5 p.p.m.), NNK (1.0 mg/kg), DBP (5000 p.p.m.) and different combinations of these toxicants 6 h/day for 16, 32 and 52 weeks. Two cytogenetic end-points, determined by the chromosomal aberration (CA) and supravital micronucleus (SMN) assays, were investigated in vivo. Our results show that all treated groups of both sexes showed genotoxic effects when compared with the control group. Additive and/or synergistic responses were observed in the CA assay for all test periods when mice of both sexes were exposed to ozone and NNK, ozone and DBP and the combination of ozone, NNK and DBP. In the SMN assay, additive interactions were noted for both sexes in the 16 and 32 week studies, similar to the results with the CA assay. All combination groups of both sexes showed synergistic interactions in the 52 week study. The results indicate that combined exposure to ozone, NNK and DBP in both sexes of mice has enhanced genotoxic effects compared with exposure to ozone alone.     

Assessment of environmental and occupational exposures to butadiene as a model for risk estimation of petrochemical emissions

1,3-Butadiene (BD) is an important industrial chemical and environmentalcontaminant, e.g. in urban air, traffic exhausts and tobaccosmoke. It has been shown to be genotoxic in vitro and in vivoand carcinogenic in rodents, mice being more sensitive thanrats. The present study confirmed this species difference. Usingmicronuclei in erythrocytes or bone marrow as a marker, miceresponded at an effective level of 50 p.p.m., while the highestineffective level in rats was 500 p.p.m. (inhalation of BD for5 days). A dose-dependent increase in N-terminal valine haemoglobinadducts was seen in both rats and mice, but the adduct levelsin the latter species were on average five times higher. Forthe first time, specific N⁶-alkyldeoxyadenosine adducts wereidentified in lung and liver DNA of rats exposed to BD by inhalation.No significant difference in DNA adduct level was seen in lungtissue of rats and mice at similar exposure levels. Occupationalexposure levels to BD in the European Process industry are variable,but generally <1 p.p.m. Haemoglobin adduct levels were seento be increased among the worker groups with higher potentialexposure to BD (process work, bomb voiding and repair duties)as compared with adduct levels in less exposed workers in maintenanceand the laboratory or control personnel. However, the N-terminalvaline haemoglobin adducts measured in the workers were oneto two orders of magnitude lower than extrapolated for the sameexposure dose in mice. In the same workers no exposurerelatedeffects were seen in the cytogenetic parametres studied, i.e.chromosomal aberrations, sister chromatid exchanges or micronucleiin peripheral blood lymphocytes, or in the Ras oncoprotein levelsof plasma samples. The studies so far conducted suggest thathuman exposure at the levels seen in the present day processindustry can be documented at the biological dose level usinghaemoglobin adduct measurement, but not at the biological effectlevel using cytogenetic biomarkers. In order to quantitate thehuman genotoxic risk of BD exposure more work needs to be doneon the role of other active BD metabolites than l,2-epoxy-3-buteneand on the genetic polymorphisms controlling the variabilityof individual responses. ⁶To whom correspondence should be addressed     

Methyl isocyanate: an evaluation of in vivo cytogenetic activity

The ability of inhaled methyl isocyanate (MIC) to induce genotoxic and cytotoxic damage in vivo was evaluated by assessing the induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in bone marrow metaphase cells, the induction of micronuclei in polychromatic erythrocytes (MN-PCEs), and the inhibition of bone marrow cellular proliferation and erythropoiesis. B6C3F1 mice were exposed to MIC by two exposure regiments: in two experiments, male mice only were exposed to 3, 10, and 30 ppm for 2 hr; in four experiments, male and female mice were exposed to 1 and 3 ppm (in one experiment, to 6 ppm, also), 6 hr per day for 4 consecutive days. The various cytogenetic endpoints were analyzed in bone marrow and peripheral blood (4-day exposure regimen only) samples taken from bromodeoxyuridine tablet-implanted animals killed 11 to 22 hr after cessation of the exposure to MIC. Exposure to MIC for 2 hr induced a significant delay in cellular proliferation but did not induce a significant increase in CAs, SCEs (evaluated at 3 and 10 ppm, only) or in bone marrow MN-PCEs. Also, this exposure regimen did not inhibit the rate of erythropoiesis. Following exposure to MIC for 4 days, a weak but significant increase in CAs and SCEs was observed in male (in one experiment) and in female (in two experiments) mice. The induction was especially apparent in the single experiment in which mice were exposed to 6 ppm MIC. At this concentration, a significant increase in MN-PCEs in peripheral blood was observed in male but not female mice. Delay in bone marrow cell proliferation was observed in male mice beginning at 3 ppm and in female mice at 6 ppm. The 4-day exposure regimen resulted also in a depressed rate of erythropoiesis, with male mice appearing to exhibit greater depression than female mice. The results demonstrate that exposure to MIC by inhalation results in bone marrow damage, indicating the systemic genotoxic/cytotoxic activity of MIC and/or reactive metabolites.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Induction of micronucleated erythrocytes by recombinant human erythropoietin

Induction of micronucleated erythrocytes by a recombinant humanerythropoietin (rhEPO) was examined using both in vitro andin vivo test systems. A small significant and dose-related increasein the frequency of micronucleated polychromatic erythrocytes(MNPCE) in bone marrow of mice administered i.p. with 12 500–50000 IU/kg rhEPO was induced at 48 h sampling time. A clear positivedose-response relationship and significant increase in the frequencyof micronucleated reticulocytes (MNRET) in peripheral bloodof mice administered i.p. with 400–50 000 IU/kg rhEPOwas noted at 48, 72 and 96 h sampling times. Conversely, inbacterial reverse mutation tests, no noticeable increase ofauxotrophic revertants was observed in Salmonella typhimurium,TA100, TA98, TA1535, TA1537, or Escherichia coli WP2 uvrA^–,by treatment with 188–6000 IU/plate of rhEPO, with orwithout S9 mix. Furthermore, rhEPO at 750–6000 m/ml didnot induce chromosomal aberrations in vitro in CHL cells orhuman peripheral blood lymphocytes in a direct method nor ina metabolic activation method. Moreover, chromosomal aberrationswere not detected in bone marrow cells of CD-1 male mice, evenat high rhEPO concentrations (100 000 IU/kg) in vivo. Consequently,it was concluded that errors in the process of enucleation ordifferentiation of the erythrocytes should be equally consideredas possible mechanisms for the increased frequencies of MNPCEand MNRET alongside induction of DNA damage or errors in theprocess of DNA repair.     

Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3

This study showed that non-MHC genes common to (DBA/2 H-2^d)and (DBA/1 H-2^q) gave rise to suppressor T (T_a) cells in thehybrid F₁ mice between C57BL/6 (B6) strain in the antl-FBL-3tumor responses. FBL-3, a Friend virus-induced tumor cell lineof B6 mouse origin, is highly immunogenic as shown by findingsthat syngenelc and hybrid F₁ mice with several other inbredstrains rejected up to 3 x 10⁷ tumor cells inoculated s.c. andgenerated potent CTL responses after mixed lymphocyte tumorcell culture. In contrast to these mice, (B6 x DBA/2) and (B6x DBA/1)F1 mice did not reject the tumor as the tumor dosesincreased. Progressive tumor growth in these F₁ mice was blockedby an I.p. Injection of cyclophosphamlde (250 mg/kg) on day10, but not on day 5, after tumor cell inoculation. Antl-CD4(GK1.5) mAb exerted similar therapeutic effects against tumorwhen given twice, between day 0 and 10, whereas the additionalinjection of antl-CD8 mAb enhanced the tumor growth in micethat otherwise rejected the tumor. Thus, In the response of(B6 x DBA/2)F, mice to FBL-3 tumor cells, CD4⁺ T₈ seemed todown-regulate the immunologically mediated regression of thetumor produced by CD8⁺ CTL. This was evidenced by limiting dilutionculture analyses, which showed that the frequency of an FBL-3-speclflcCTL precursor in the (B6 x DBA/2)F1 mice that rejected the tumorwith antl-CD4 mAb was 7- to 9-fold higher than that in micein which the tumor regressed spontaneously. That more than onegene was involved in suppressor T cell induction was shown bythe tumor growth pattern in (B6 x DBA/2)F₁ x B6 backcross andB6D2F2 mice.     

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.     

Cytogenetic biomonitoring in a Spanish group of agricultural workers exposed to pesticides

Sister chromatid exchanges (SCE) and chromosome aberrations(CA) were studied in the lymphocytes of 70 male agriculturalworkers occupationally exposed to several pesticides and 69matched controls, without indication of exposure to pesticides,from ‘El Maresme’ (Barcelona, Spain). Comparisonbetween both groups revealed that the individuals exposed topesticides show substantial clastogenic effects in their lymphocyteswithout Indication of increases in the basal frequency of SCE;moreover, these effects seem to be additive, increasing withthe duration of exposure measured in years. When two confoundingfactors such as age and smoking habits are considered, we foundthat these factors increase significantly the expression ofSCE although no effect was detected in the expression of CA. ¹To whom correspondence should be addressed     

Mutagenicity of an organophosphate insecticide acephate--an in vivo study in chicks

The chromosome aberration assay (CA) in bone marrow cells andthe micronucleus test (MNT) in both bone marrow and peripheralblood erythrocytes have been carried out for the evaluationof the clastogenic potential of acephate (asataf) in a chickin vivo test system. Technical grade acephate was administeredto evaluate dose-responses (25, 50 and 100 mg/kg), route-responses(i.p. and p.o.) and time-responses (6, 24 and 48 h). A comparisonof CA frequencies after acute and chronic dosing was also performed.Only 50 mg/kg of acephate induced significant bone marrow chromosomeaberrations after 24 h exposure while all three doses inducedsignificant increases in micronuclei in both bone marrow andperipheral blood erythrocytes in i.p. route only. The presenteddata confirm our earlier reports that the neonatal chicken testsystem is a convenient model and can be used as an alternativeto mammalian systems for screening some environmental contaminantsfor genotoxicity. ¹To whom correspondence should be addressed