Micronucleus formation induced by the combination of low doses of X-rays and antineoplastic drugs in bone marrow of male mice.

People are widely exposed during their lifetime to many biological, chemical, and physical agents in the environment and at work. In this paper the effects of combined exposures of nonmutagenic doses of X-rays and anticancer agents (cyclophosphamide, mitomycin C, and vinblastine) have been investigated on the induction of micronuclei in the bone marrow of laboratory mice. The combination of X-rays and anticancer drugs enhanced the frequency of micronuclei in some cases. The strongest effects were found after the combination of X-rays and cyclophosphamide at 24 h and 72 h. The combined treatment of X-rays and mitomycin C enhanced the mutagenic effect at 72 h. The combination of X-rays + vinblastine slightly potentiated the mutagenic effect at 24 h and 48 h.

Induction of micronuclei in bone marrow and sperm head abnormalities after combined exposure of mice to low doses of X-rays and acrylamide.

People come into contact with chemical and physical agents which are present in the environment and in workplaces. We investigated the effects of combined exposures to low doses of X-rays (0.05-0.25 Gy) and acrylamide (AA; 75 mg/kg bw) in the somatic and germ cells of outbred male mice by using a bone-marrow micronucleus test and a sperm morphology test. Combined treatment of germ cells to 0.25 Gy of X-rays + 75 mg/kg bw of acrylamide enhanced the effect induced by each agent given alone. The results confirmed the sensitivity to damage of spermatozoa and late spermatids, which can be demonstrated by sperm head abnormalities and reduced fertility. The sensitivity of somatic cells to acrylamide alone was similar to that of germ cells. Combined exposure to 0.05 Gy + 75 mg/kg bw of AA induced micronuclei in polychromatic erythrocytes of bone marrow although each dose did not produce a mutagenic effect. Teratogenesis Carcinog. Mutagen. 20:133-140, 2000.

[Lectures from the tutorial courses at NATO--Advanced Study Institute pt. "Human biomonitoring after environmental and occupational exposure to chemical and physical agents" (Turkey, 9/23-10/3/1999)]. / Sprawozdanie ze szkolenia naukowego NATO-Advanced Study Institute pt. "Human biomonitoring after environmental and occupational exposure to chemical and physical agents" (Turcja, 23.09-3.10.1999).

The NATO Science Programme joining in the celebration of 50th Anniversary of Founding of the NATO by organisation of NATO Advanced Study Institute "Human Monitoring after Environmental and Occupational Exposure to Chemical and Physical Agents", which was held in Tekirova-Antalya (Turkey), September 23-October 3, 1999. The director of ASI was dr Diana Anderson from TNO-BIBRA (UK). The members of Scientific Organizing Committee were also dr R. Sram (Czech Republik), dr A. Karakaya (Turkey), Dr P. O'Neill (USA), dr R. Bos (Netherlands), dr M. Lotti (Italy). It was a high-level tutorial course for scientists at the post-doctoral level from NATO countries and from NATO Cooperation Partner countries. NATO-ASI attended about 100 scientists from about 30 countries. There were 40 lectures, 20 oral presentations and 43 posters presented, 19 authors of posters were invited to additional short oral presentations. Subject of course concerned undesirable effects of chemical and physical agents on human health. The aim of NATO-Advanced Study Institute was the meeting of scientists working in different fields of science to present and discuss the knowledge and recent developments in the field of human monitoring. The majority of lectures concerned about biomonitoring of people exposed to genotoxic agents at work place and environment. Dr A. Autio (Switzerland) presented definitions of different kinds of bimarkers proposed by the Committee on Biological Markers in Environmental Health of USA Academy of Science/National Research Council. Dr D. Anderson (UK) introduced history of biomonitoring. The main lecturers on this topic were dr W. Au (USA), dr R. Sram (Czech Republik), dr M. Lotti (Italy), dr J. Timbell (USA), Dr E. Moustacchi (France). The following group of lectures presented by dr D. Anderson (UK), dr A. Wyrobek (USA), dr J. Bonde (Dennmark), dr H. Norppa (Finland) was regarded to male-mediated mutagenic effect in offspring induced by genotoxic physical and chemical agents. This part of course was the most interesting to the author of this report. She has presented the poster "Male-mediated F1 effects in mice subchronic exposed to low doses of X-rays". The author of this report found NATO-ASI as very fruitful initiative for scientific view-exchange between scientists from NATO countries and for NATO Cooperation Partner countries.

[Biological effect of exposure to butadiene]. / Biologiczne skutki ekspozycji na butadien.

People can be exposed to 1,3-butadiene in work place (rubber industry) as well as in natural environment (car exhausts fumes, cigarette smoke). Butadien on its own is not genotoxic, but is metabolized to mutagenic and carcinogenic epoxydes, 1,2,3,4-diepoxybutane and 3,4-epoxybutene in the organism of mammals and human. 1,3-butadiene has been shown to be a potent carcinogen in animals and human. Laboratory investigations showed also toxic and mutagenic abilities of butadiene and its metabolities. Interspecies differences in sensitivity to butadiene are caused by differences in metabolic transformations of butadiene in different species.

Induction of micronuclei in mouse bone marrow after combined X-rays-cyclophosphamide and X-rays-mitomycin C treatments.

The induction of micronuclei in polychromatic erythrocytes of bone marrow of Pzh:SWISS mice after combined treatment with X-rays and cyclophosphamide (CP) or X-rays and mitomycin C (MMC) were investigated. Combinations of high (1.00 Gy + 100 mg/kg bw CP and 1. 00 Gy + 5.25 mg/kg bw MMC) and low (0.25 Gy + 25 mg/kg bw CP and 0. 25 Gy + 1.75 mg/kg bw MMC) doses were used. Both chemicals enhanced the mutagenic effects caused by irradiation. After combined treatment with high doses of X-rays + CP and X-rays + MMC at different sample times increases in frequency of micronuclei were observed. Mutagenic effects were found also after treatment with two low doses, when irradiation alone produced no effects. The effects of combined treatments are generally similar to the additive effect of the single treatments.

[Morphological changes in mice sperm head after exposure to radiation and to cyclophosphamide]. / Zmiany morfologiczne mysich plemników po skojarzonym dzialaniu promieniowania X i cyklofosfamidu.

Male mice Sfis:Pzh were exposed to X-rays, cyclophosphamide or combination of both agents. Each of agent was given in low (0.25 Gy, 25 mg/kg bw CP) or high (1.00 Gy, 100 mg/kg bw CP) doses. Germ cells were exposed to agents as spermatogonia. After 35 days sperm abnormalities test was performed. Exposure to one of agents only, did not enhance statistically significant frequency of morphologically abnormal spermatozoa. Combined treatment of spermatogonia to both agents in low as well in high doses induce clear biological effects, but only combination of high doses (1.00 Gy + 100 mg/kg bw CP) induce statistically significant effect. Results obtained in this study confirmed, that ability of different agents to induce sperm-shape abnormalities is related to its ability to induce mutations in germ cells.

Flavonoids modulate comet assay responses to food mutagens in human lymphocytes and sperm.

The flavonoids, silymarin, myricetin, quercitin, kaempferol, rutin and kaempferol-3-rutinoside have been examined in combination with the food mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-3-methylimidazo-(4,5-f) quinoline (IQ), in the Comet assay in human lymphocytes from donor A and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in lymphocytes and sperm. The assays were performed in the absence of metabolic activation, since when quercetin and kaempferol were examined in blood with metabolic activation, there was little or no difference in response to that obtained in its absence. In the blood, there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm, with silymarin, myricetin and quercitin, antigenotoxic effects only were observed, but with kaempferol, in general, there were no protective effects. The food mutagen, 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), was also examined in addition to Trp-P-2 and IQ in combination with silymarin and myricetin in donors A and C in human lymphocytes only. Similar exacerbation of effects were found at low doses of these flavonoids with antigenotoxic effects at high doses. This was confirmed in the Ames test. There were slightly different profiles in lymphocytes and sperm, but antigenotoxic effects were observed over a similar dose range. This would suggest that effects occur in somatic and germ cells on a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

Evaluation and characterization of micronuclei in early spermatids of mice exposed to 1,3-butadiene.

The frequency of micronuclei induced in mouse meiotic cells after exposure to 1,3-butadiene has been evaluated in early spermatids. Germ cells were isolated from mice exposed to three butadiene concentrations (130, 250 and 500 ppm), at time intervals allowing to evaluate effects induced in late spermatocytes or at the stage of prelepotene/differentiating spermatogonia. The characterization of the origin of micronuclei, by simultaneous detection of centromeric and telomeric sequences, was also done on spermatid preparations from the 250 ppm concentration. The same analysis was carried out on a group of mice treated with the major butadiene metabolite, 1,2,3,4-diepoxybutane. The results obtained indicate a weak clastogenic effect of butadiene to premeiotic germ cells in the mouse.

Effect of various genotoxins and reproductive toxins in human lymphocytes and sperm in the Comet assay.

There have been conflicting reports as to whether the mean sperm count in some men has diminished over the last 50 years. The downward trend has been suggested to coincide with an increase in exposure to estrogen-like compounds. These estrogenic substances are ubiquitous in the environment. We have examined the effect of such substances (diethylstilbestrol, beta-estradiol, daidzein, genestein, and nonylphenyl) in the single cell gel electrophoresis assay (Comet assay) in human sperm and compared responses with those from human peripheral lymphocytes in the same donor and in peripheral lymphocytes from a female donor. In addition, effects from the estrogens have been compared to those from known reprotoxins and genotoxins. These include lead sulfate, nitrate and acetate, dibromochloropropane, ethylene glycol monoethyl ether, 1,2-epoxybutene, and 1,2,3,4-diepoxybutane. All compounds produced positive responses, but ethylene glycol monoethyl ether only produced positive responses in sperm cells in the male and not in peripheral lymphocytes, and similarly the phytoestrogens (genistein, daidzein) were less responsive in the peripheral lymphocytes in the male than in the sperm. This may be due to greater sensitivity of sperm cells because of their lack of repair. However, since damage was generally seen over a similar dose range, a one-to-one ratio of somatic and germ cell damage was observed and has implications for man for risk assessment purposes.

Modulating effects of flavonoids on food mutagens in human blood and sperm samples in the comet assay.

The flavonoids silymarin, myricetin, quercetin, kaempferol, rutin, and kaempferol-3-rutinoside have been examined in combination with the food mutagens 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp) and 2-amino-3-methylimidazo-4,5-f)quinoline (IQ) in the Comet assay in human lymphocytes from donors A and B and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in human lymphocytes and sperm over a similar dose range in the absence of metabolic activation. Only quercetin and kaempferol were examined in blood with metabolic activation, but there was no difference in response to that obtained without activation. In the blood there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm this was also the case with silymarin and myricetin. With kaempferol there was no antigenotoxic effect and quercetin protected below baseline levels. Since the effects were observed in lymphocytes and sperm over a similar dose range, it would suggest that the Comet assay responses occur in somatic and germ cells in a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

DNA integrity in human sperm.

Since the 1970s there have been conflicting reports of decreasing sperm counts in man and increasing testicular cancer. There is a hypothetical link between apparent adverse trends in several measures of human reproductive health and exposure to endocrine disrupters. Rodent bioassays are not suited for the large-scale screening of such chemicals because of their costs, complexity, and ethical concerns. Various in vitro assays have been used to examine the effects of these chemicals, but none has directly used semen as one of the target tissues in man. The present study has examined in the alkaline Comet assay in human sperm the effect of two estrogens--beta-estradiol and the phytoestrogen daidzein--and 1,2-epoxybutene, a metabolite of 1,3-butadiene, and compared them with the effects of the known reprotoxin, dibromochloropropane, in two fertile and two infertile frozen sperm samples and two fresh fertile samples. While differences were detected in the frozen fertile and infertile samples with flow cytometry, in the Comet assay both frozen and fresh samples exposed to the chemicals in vitro from fertile and infertile men produced similar altered responses by comparison with untreated samples. The integrity of DNA is necessary not only for the noncancerous state, but also for the accurate transmission of genetic material to the next generation. Thus this assay may be useful for examination of chemicals in fresh and frozen sperm samples.

Effects in the Comet assay of storage conditions on human blood.

The Comet assay is a rapid and sensitive method for analyzing single cells for DNA damage. Using human lymphocytes, the assay is particularly useful for human monitoring studies, as well as for in vitro genotoxicity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood is taken. It would be useful if some samples could be stored and examined at a different time, without loss of viability or other factors affecting responses. It is thus important to understand the effects of storage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two laboratories, blood was taken from various donors and stored under different conditions. It was examined on day 1 (day on which sample was taken) and days 2, 3, 4, 5, or 8 at room temperature, 4 degrees C, or -20 degrees C. Cells were treated after storage (from day 2 onward) with bleomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed either by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liverpool UK. Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There was no loss of cell viability at 4 degrees C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest that on days 1-4 for the untreated samples at room temperature or 4 degrees C there were no biologically meaningful changes in both the different categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4 degrees C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less variability between samples at 4 degrees C than at room temperature in one laboratory. The reverse was true in the other. This would suggest that samples can probably be stored up to day 4 at 4 degrees C or room temperature without any untoward effects. Provided samples can be processed within this 4-day time frame, it would not seem necessary to cryopreserve samples at -196 degrees C.

Mouse dominant lethal and sperm abnormality studies with combined exposure to X-rays and mitomycin C.

The induction of dominant lethal effects and sperm abnormalities in Pzh:Swiss male mice after treatments with X-rays and mitomycin C (MMC) was investigated. Combinations of high (1.00 Gy + 5.25 mg/kg bw MMC) and low (0.25 Gy + 1.75 mg/kg bw MMC) doses of both agents were used. Exposure to high doses of X-rays + MMC induced an increased rate of dominant lethal mutations in spermatogonia and late spermatocytes. Combined treatment with low doses of X-rays and MMC was not mutagenic in any stage of spermatogenesis. MMC increased the frequency of abnormal spermatozoa after exposure alone and in combination with X-rays. Treatment with two high doses (1.00 Gy + 5.25 mg/kg bw MMC) induced 58.4% abnormal spermatozoa. After combined exposure to low doses of both agents 35.7% spermatozoa with malformations were observed.