Induction Of Somatic Mutations Research Articles

In vivo evaluation of the toxic activity and genotoxicity of the Hymenaea courbaril L.'s resin in Drosophila melanogaster.

Due to the negative consequences carried by the usage of synthetic insecticides, a global interest into finding substitutes for these chemical compounds through natural products has arisen. When yielded to external attacks, plants generally produce metabolites to defend themselves. The physicochemical characteristics of this kind of compounds have allowed their usage as potential bioinsecticides. The Hymenaea courbaril L. (algarrobo) has proven to be a plant rich in metabolites with outstanding biological activity, in such a way that some of its extracts have been tested as insecticides. The goal of this study was to know the phytochemical composition of Hymenaea courbaril L.’s resin and perform evaluations in vivo of its toxic and genotoxic effects in the biological model Drosophila melanogaster. For this, two resin extracts were prepared and both a phytochemical analysis were carried out on them, having found in the ethanolic total extract the presence of terpenes, flavonoids and coumarins, while in the partial ethanolic extract only presence of terpenes and flavonoids was found. Drosophila larvae were submitted to different concentrations of the extracts and both the survival and the sexual ratio were evaluated, finding that larvae are more sensitive to the partial ethanolic extract. Subsequently, the induction of somatic mutation and mitotic recombination (SMART) was evaluated in the flies’ eyes. The most significant affectations at a genotoxic level were found when larvae were tested with the partial extract, indicating that possibly the coumarins absence makes this insect more susceptible to damages at a genetic material level.

Induction of somatic mutations by low concentrations of tritiated water (HTO): evidence for the possible existence of a dose-rate threshold.

Tritium is a low energy beta emitter and is discharged into the aquatic environment primarily in the form of tritiated water (HTO) from nuclear power plants or from nuclear fuel reprocessing plants. Although the biological effects of HTO exposures at significant doses or dose rates have been extensively studied, there are few reports concerning the biological effects of HTO exposures at very low dose rates. In the present study using a hyper-sensitive assay system, we investigated the dose rate effect of HTO on the induction of mutations. Confluent cell populations were exposed to HTO for a total dose of 0.2 Gy at dose rates between 4.9 mGy/day and 192 mGy/day by incubating cells in medium containing HTO. HTO-induced mutant frequencies and mutation spectra were then investigated. A significant inflection point for both the mutant frequency and mutation spectra was found between 11 mGy/day and 21.6 mGy/day. Mutation spectra analysis revealed that a mechanistic change in the nature of the mutation events occurred around 11 mGy/day. The present observations and published experimental results from oral administrations of HTO to mice suggest that a threshold dose-rate for HTO exposures might exist between 11 mGy/day and 21.6 mGy/day where the nature of the mutation events induced by HTO becomes similar to those seen in spontaneous events.

Intraperitoneal sodium metavanadate exposure induced severe clinicopathological alterations, hepato-renal toxicity and cytogenotoxicity in African giant rats (Cricetomys gambianus, Waterhouse, 1840).

Pollution of environment due to increased exploitation of minerals has been on the rise, and vanadium, a metal in the first transition series essential for mammalian existence, is a major component of air pollution. This study investigated the clinico-pathological, hepato-renal toxicity, and cytogenotoxicity of intraperitoneal exposure of African giant rats (AGRs), a proposed model for ecotoxicological research to sodium metavanadate. A total of 27 adult male African giant rats weighing 975 ± 54.10g were distributed into two major groups: sodium metavanadate (SMV) treated and control. They were observed daily for clinical signs of toxicity. Four rats from each group were randomly collected and sacrificed after 3, 7, and 14days of SMV treatment. Liver, kidney, and bone marrow were analyzed for histopathology and micronucleated normochromated and polychromated erythrocytes (MNNCE and MNPCE), respectively. Clinical signs in treated AGR include sluggish and weak movements, un-groomed fur, and labored breathing. Histology of the kidney revealed severe glomerular atrophy, tubular ectasia, and vacuolar degeneration of tubular epithelium, while liver histology showed sinusoidal congestion and severe hepatocellular necrosis after 14days SMV exposure. Also, MNNCE and MNPCE significantly increased with a decrease in PCE/NCE ratio in SMV-treated AGR, suggestive of alternations in bone marrow cell proliferation. Hence, SMV treatment to AGR resulted to severe clinicopathologic alterations, kidney, and liver dysfunction and cytogenotoxicity evident by somatic mutation induction which could be severe with prolonged exposure. This suggests African giant rat as an ecotoxicological model to measure major health risks to animals and human populations in highly polluted environment.

Induction of somatic mutations by low-dose X-rays: the challenge in recognizing radiation-induced events.

It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0.15 Gy. In contrast, mutant frequencies at doses of <0.1 Gy occurred at non-significant levels. The mutation spectrum in HPRT-deficient mutants revealed that the type of mutations induced by low-dose exposures was similar to that seen in spontaneous mutants. An apparent change in mutation type was observed for mutants induced by doses of >0.2 Gy. Our observations suggest that there could be a critical dose for mutation induction at between 0.1 Gy and 0.2 Gy, where mutagenic events are induced by multiple DNA double-strand breaks (DSBs). These observations also suggest that low-dose radiation delivered at doses of <0.1 Gy may not result in DSB-induced mutations but may enhance spontaneous mutagenesis events.

Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers

Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0–75Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20Gy gamma rays. Irradiated shoot tips were sub-cultured and M1V1–M1V3 generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20gl−1 PEG6000. Of the M1V3 plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20Gy gamma radiation and regenerated on selective culture media containing 10gl−1 PEG6000 concentration, and the second cluster was further divided into five sub-clusters.

Relationships between cisplatin-induced adducts and DNA strand-breaks, mutation and recombination in vivo in somatic cells of Drosophila melanogaster, under different conditions of nucleotide excision repair

Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC–ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/ w + SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G–G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G–G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10 4 cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences.

Radiation induced cancer arises from a somatic mutation

Experimental data from the literature are presented to create a chain which links radiationinduced cancer in animals to cell reproductive death and then to chromosomal aberrationsand somatic mutations. The cancer data reveal the same peak value of cancer inductionfollowing different radiation exposures which leads to an association between cancerinduction and cell killing. Other data show a direct correlation between cell survival andchromosomal aberration yield independent of whether a sensitiser is used or not. Dataon the induction of somatic mutations in mammalian cultured cells show thesame direct relationship between mutation frequency and cell killing followingneutron and gamma ray exposures. Taken as a whole, the experimental dataprovide convincing evidence that radiation induced cancer arises from chromosomaldamage.

Two Major Factors Involved in the Reverse Dose-rate Effect for Somatic Mutation Induction are the Cell Cycle Position and LET Value

To study mechanisms which could be involved in the reverse dose rate effect observed during mutation induction after exposure to high LET radiation, synchronized mouse L5178Y cells were exposed to carbon 290 MeV/n beams with different LET values at the G2/M, G1, G1/S or S phases in the cell cycle. The frequency of Hprt-deficient (6-thioguanine-resistant) mutant induction was subsequently determined. The results showed that after exposure to high LET value radiation (50.8 and 76.5 keV/microm), maximum mutation frequencies were seen at the G2/M phase, but after exposure to lower LET radiation (13.3 keV/microm), the highest mutation frequencies were observed at the G1 phase. The higher LET beam always produced higher mutation frequencies in the G2/M phase than in the G1 phase, regardless of radiation dose. These results suggest that cells in the G2/M phase is hyper-sensitive for mutation induction from high LET radiation, but not to mutation induction from low LET radiation. Molecular analysis of mutation spectra showed that large deletions (which could include almost entire exons) of the mouse Hprt gene were most efficiently induced in G2/M cells irradiated with high LET radiation. These entire exon deletions were not as frequent in cells exposed to lower LET radiation. This suggests that inappropriate recombination repair might have occurred in response to condensed damage in condensed chromatin in the G2/M phase. In addition, by using a hyper-sensitive mutation detection system (GM06318-10 cells), a reverse dose-rate effect was clearly observed after exposure to carbon beams with higher LET values (66 keV/microm), but not after exposure to beams with lower LET values (13.3 keV/microm). Thus, G2/M sensitivity towards mutation induction, and the dependence on radiation LET values could both be major factors involved in the reverse dose rate effect produced by high LET radiation.

Prevalence of BER gene polymorphisms in sporadic breast cancer

Studies suggest that breast cancer is initiated by the induction of somatic mutations from errors in the base excision repair (BER) of endogenous estrogen-induced abasic sites. If so, the inheritance of certain polymorphic mutations in BER genes involved in the incorporation and management of such errors should increase the risk of breast cancer. To test this hypothesis, we examined breast tissues from 48 women (controls, histopathologically normal tissue from reduction mammoplasty) and 40 women with breast cancer (breast tumor-adjacent, histopathologically normal tissues) for the presence of reported polymorphic mutations in four BER genes. The breast tissues were obtained from the Cooperative Human Tissue Network-western division and from the University of Nebraska Medical Center. Using PCR-RFLP procedures, the XRCC1 gene was examined for Arg194Trp and Arg399Gln, APE1 for Asp148Glu, LIG3alpha for Arg780His and PARP1 for Pro377Ser mutations. The women in this study carried only the XRCC1 Arg399Gln polymorphism. This result was surprising because APE1 148Glu was reported to be frequently inherited (allele frequency, 0.47-0.495) by USA and European women. Thus, the USA women in our study are genetically different from those in the previous studies. Among the control women, 21 (43.75%) were Arg/Arg wild-types, 20 (41.67%) were Arg/Gln heterozygotes and 7 (14.6%) were Gln/Gln homozygotes. Among the breast cancer cases, 11 (27.5%) were Arg/Arg wild-types, 16 (40%) were Arg/Gln heterozygotes and 13 (32.5%) were Gln/Gln homozygotes. Thus, the Gln allele was significantly more frequent in breast cancer cases (allele frequency, 0.52) than in controls (allele frequency, 0.35), suggesting that XRCC1 399Gln may enhance the risk of breast cancer.

LET dependence for somatic mutation induction by heavy ion beam with low-dose rate

LET dependence for somatic mutation induction by heavy ion beam with low-dose rate

The effect of transposon P(GUS . p53.259H)on the frequency of tumor clones in Drosophila melanogaster wtsp2/+ heterozygotes

We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.

In vitro induction of somatic mutations in human peripheral B lymphocytes.

The major cellular and molecular factors that lead to the induction of somatic mutations in human B cells remain unclear. This study describes an approach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism analysis on the descendant cultures we were able to demonstrate that the Ig gene of B cells was induced to mutate. Three clones were isolated from a single cell culture of naive B cells immunized against the semi-conserved region of V3 of HIV-1, which contained two point mutations at the CDR2 region leading to amino acid codon change. Two clones were also obtained from a single cell culture of memory B cells immunized against the recall antigen tetanus toxoid. These clones showed one point mutation at the CDR1-FR2 border region leading to amino acid codon change. Fab constructs from the mutated culture of naive B cells immunized against V3 were cloned to a procaryotic expression vector. The expression products of two clones showed anti-V3 specificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through specific interaction with antigen and antigen-activated T cells.

Induction of somatic mutations but not methylated DNA adducts in λlacZ transgenic mice by dichlorvos

In order to examine the in vivo genotoxic activity of dichlorvos, λlacZ transgenic mice (Muta™Mouse) were treated i.p. with single (4.4 or 11 mg/kg) or multiple (5×11 mg/kg) doses of this agent and sacrificed 4 h or 14 days post-treatment for DNA adduct measurement or mutant frequency analysis, respectively. Neither methylated DNA adducts nor an increase in mutant frequency were detected in the bone marrow, white blood cells, liver, spleen, lung, brain and sperm cells after the single doses. However, following multiple dosing a statistically significant 3-fold increase in mutant frequency was observed in the liver, while a non-statistically significant increase was observed in the bone marrow. In contrast, dimethylsulphate, a model methylating agent, gave rise to detectable DNA adducts but no increase in mutant frequency following i.p. administration of single (30 mg/kg) or multiple (10×6 mg/kg) doses.

Carcinogen exposure, p53 alteration, and K-ras mutation in synchronous multiple primary lung carcinoma

Synchronous multiple primary lung tumors (SMPLT) have been estimated to occur in 1% of lung carcinoma patients. Criteria for SMPLT diagnosis include different cancer histologies, location in different lobes of the lung, or genetic discordance. Patients with SMPLT have a poor clinical prognosis with decreased 5-year disease survival, despite diagnosis at an early stage. Field cancerization (the induction of somatic mutation in large clones of epithelial cells after carcinogen exposure) has been proposed to be an integral process involved in the development of SMPLT. Host factors also may contribute to the development of SMPLT. The authors investigated the occurrence of p53 and K-ras alterations in tumors from SMPLT patients and also studied the association between carcinogen exposure and polymorphic metabolic traits in SMPLT patients, comparing these patients with individuals with one primarily lung tumor. In a case-control study of lung carcinoma susceptibility, 6 patients were identified whose 33 multiple tumors met the criteria of SMPLT. The incidence was 3.6% (16 of 451 patients), which was higher than many previously published series. Among the multiple tumors, 73% (24 of 33 tumors) were adenocarcinomas. Patients with SMPLT smoked more (73.0 pack-years vs. 56.2 pack-years; P = 0.07) and longer (45.8 years vs. 37.0 years; P < 0.03) than patients with only 1 tumor. For those patients who stopped smoking, patients with SMPLT had stopped smoking more recently than those with a single primary tumor (3.4 years vs. 7.3 years; P = 0.08). A total of 39% of SMPLT tumors (13 of 33 tumors) had detectable p53 alterations; 36% had genetic changes in p53 measured by polymerase chain reaction-single strand conformation polymorphism, and 33% showed positive immunostaining for p53 protein. This was comparable to the occurrence of p53 mutations and immunostaining in single tumor cases (30%). Age, gender, family history of cancer, and the prevalence of polymorphic metabolic traits previously associated with lung carcinoma susceptibility did not differ among SMPLT patients compared with patients with a single tumor. The patients with SMPLT had significantly more tobacco exposure, and their tumors apparently had independently arising p53 and K-ras mutations, suggesting that field cancerization may be important in lung carcinogenesis.

Induction of Ig Somatic Hypermutation and Class Switching in a Human Monoclonal IgM+ IgD+ B Cell Line In Vitro: Definition of the Requirements and Modalities of Hypermutation

Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities.

RBE-LET relationships of high-LET radiations in Drosophila mutations.

The relative biological effectiveness (RBE) of 252Cf neutrons and synchrotron-generated high-energy charged particles for mutation induction was evaluated as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-color in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye-color is due to an intragenic structural change (2.96 kb-DNA excision) in the white locus on the X-chromosome. The measurements were performed in a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clone can be detected simultaneously in the same individual. Larvae were irradiated at the age of 3 days post oviposition with 252Cf neutrons, carbon beam or neon beam. For the neutron irradiation, the RBE values for wing-hair mutations were larger than that for eye-color mutation by about 7 fold. The RBE of carbon ions for producing the wing-hair mutations increased with increase in LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair. For neon beam irradiation, the RBE values for wing-hair mutations peak near 150 keV/micron and decrease with further increase in LET. On the other hand, the RBE values for the induction of the eye-color mutation are nearly unity in 252Cf neutrons and both ions throughout the LET range irradiated. We discuss the relationships between the initial DNA damage and LET in considering the mechanism of somatic mutation induction.

Genetic effects of 1,3-butadiene and associated risk for heritable damage

A summary of the results of the studies conducted in the EU Project `Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites in somatic and germ cells of mice, rats and man' is presented. Results of the project are summarized on the detection of DNA and hemoglobin adducts, on the cytotoxic and clastogenic effects in somatic and germinal cells of mice and rats, on the induction of somatic mutations at the hprt locus of experimental rodents and occupationally exposed workers, on the induction of dominant lethal mutations in mice and rats, and on heritable translocations induced in mice, after exposure to butadiene (BD) or its major metabolites, butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). The primary goal of this project was to collect experimental data on the genetic effects of BD in order to estimate the germ cell genetic risk to humans of exposure to BD. To achieve this, the parallelogram approach of Sobels was employed. The presented estimates of heritable damage in man as a consequence of butadiene exposure are based on data for heritable translocations and bone marrow micronuclei induced in mice and chromosome aberrations observed in lymphocytes of exposed workers. A doubling dose for heritable translocations in human germ cells of 4900 ppm/h is estimated, which, assuming cumulative BD exposure over the sensitive period of spermatogenesis, corresponds to 5–6 weeks of continuous exposure at the workplace to 20–25 ppm. Alternatively, the rate of heritable translocation induction per ppm/h of BD exposure is estimated to be approximately 0.8 per million live born, compared to a spontaneous incidence of balanced translocations in humans of approximately 800 per million live born. These estimates have large confidence intervals and are only intended to indicate orders of magnitude of human genetic risk. These risk estimates are based on data from germ cells of BD-exposed male mice. The demonstration that clastogenic damage was induced by DEB in preovulatory oocytes at doses which were not ovotoxic implies that additional studies on the response of mammalian female germ cells to BD and its metabolites are needed. The basic assumption of the above genetic risk estimates is that experimental mouse data obtained after BD exposure can be extrapolated to humans. Several points exist in the present report and in the literature which contradict this assumption: (1) the level of BMO–hemoglobin adducts was significantly elevated in BD-exposed workers; however, it was considerably lower than would have been predicted from comparable rat and mouse exposures; (2) the concentrations of the metabolites DEB and BMO were significantly higher in mouse than in rat blood after BD exposure. Thus, while metabolism of BD is qualitatively similar in the two species, it is quantitatively different; (3) no increase of HPRT mutations was shown in 19 workers exposed on average to 1.8 ppm of BD, while in a different population of workers from a US plant exposed on average to 3.5 ppm of BD, a significant increase of HPRT variants was detected; and (4) data from cancer bioassays and cancer epidemiology suggest that rat is a more appropriate model than mouse for human cancer risk from BD exposure. However, the dominant lethal study in rats gave a negative result. At present, we do not know which BD metabolite(s) may be responsible for the genetic effects even though the bifunctional alkylating agent DEB is the most likely candidate for the induction of clastogenic events. Unfortunately, methods to measure DEB adducts in hemoglobin or DNA are only presently being developed. Despite these several uncertainties, the use of the mouse genetic data is regarded as a justifiable and conservative approach to human genetic risk estimation given the considerable heterogeneity observed in the biotransformation of BD in humans.

Carcinogenesis Induced by UV and Ionizing Radiation

The increase in the incidence of cancers in the general population, in particular the rapid increase of certain types of skin cancers in recent years , represents an alarming threat for human health and a permanent challenge for scientists. In fact, a doubling of the frequence of melanoma during the last fifteen years and a doubling of non melanoma skin cancers have been observed in the last ten years in France; already in the He of France one counts approximately 8000 new cases per year (Dore et al. 1990, Cesarini , personal communicat ion). The worldwide reported rapid rise in the incidence of these cutaneous malignancies (Glass and Hoover 1989) is almost entirely attributed to increased cutaneous exposure to solar UV radiation mostly related to present social behaviours including skin tanning and increased outdoor leisure activities (Mukhtar and Elmets 1996). Increased UV exposure due to stratospheric ozone depletion has also become a factor of concern (Henriksen et al. 1990) as well as indoor sunbed exposures (Autieret al. 1994. 1995). A 10% depletion in the total-column of ozone is expected to cause , if sustained over several decades , a 2 0 % increase in basa celt carcinoma and an about 4 0 % increase in squamous cell carcinoma (Van der Leun, 1995). However , at the present time the decrease in ozone layers observed cannot be taken as the main factor for the increase in skin cancer. The recent development of molecular biology has helped to get a better understanding of the mechanisms involved and to enlarge the classical concept of carcinogenesis including as principal steps the initiation and promotion of somatic cells. Actually, carcinogenesis is a multistep phenomenon (Figure 1). ln particular, one of the presently favoured hypotheses describes cancer induction as a consequence of the induction of somatic mutations following the induction of DNA damage , error prone or incomplete enzymatic repair of the lesions induced leading to altered gene expression , i.e. the inactivation of certain tumour suppressor genes and/or the activation of oncogenes which favour the progression of transformed cells and tumor development. Indeed, it is thought that the activation of genes which control cell proliferation facilitates the expansion and clonal selection of initiated and genetically instable cells showing an acceleration in cell cycle progression. If such cells escape the immunologic survey system, tumor cells may built foci and, depending on their nature (see models for the induction of melanoma (Albino and Fountain 1996) and squamous cell carcinoma (Ziegler et al. 1996)), may start spreading over the whole body in form of metastasis. That the individual repair capacities play an important role in carcinogenesis has become evident from studies on human cancer-prone diseases such as Xeroderma pigmentosum (XP) (Sarasin 1994, Kraemer 1996, Grossman and Wei 1995), Fanconi ' s Anemia (FA) (Moustacchi 1994) and Ataxia telangectasia (AT) (Aurias 1994). For example , X P patients are more than 1000 fold more susceptible to the induction of skin cancers by sunlight than normal individuals (Kraemer 1996). Ultraviolet radiation can be distinguished from low density ionizing radiation such as X-rays by differences in efficacy and specificity of the cancer induced. It appears that at comparable cytotoxic level ultraviolet radiation exerts a higher mutagenic and carcinogenic potential than ionizing radiation. This is probably due to differences in energy deposition in cellular targets and in the frequency and type of lesions induced by these agents . In physics and many industrial and medical applications, lasers are employed emitting strong monochromatic light in different regions of the electromagnetic spectrum including ultraviolet, visible and infrared radiation as well as soft X-rays and very short wave ultraviolet radiation < 200 nm . Despite the fact that these highenergy monochromatic light sources are highly

Induction of Somatic Mutation in a Human B Cell Line In Vitro

Both the B cell–surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by coculture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV H gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro–induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V HDJ H gene segment and do not show evidence of antigen-driven selection.

Induction of somatic mutation and recombination by four inhibitors of eukaryotic topoisomerases assayed in the wing spot test of Drosophila melanogaster.

Four inhibitors of eukaryotic topoisomerases were investigated for genotoxic effects in the wing spot test of Drosophila melanogaster. As a somatic mutation and recombination test (SMART) this assay assesses mitotic recombination and mutational events of various kinds. We studied camptothecin as a topoisomerase I inhibitor, as well as ellipticine as an intercalating inhibitor and teniposide and etoposide as two non-intercalating inhibitors of topoisomerase II. Wing spots were induced in flies trans-heterozygous for the recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) as well as in flies heterozygous for mwh and the multiply inverted TM3 balancer chromosome. All four compounds proved significantly genotoxic in this test. The spot induction frequencies formally standardized to the millimolar unit of exposure dose decreased in the order camptothecin > teniposide > ellipticine greater, similar etoposide in the mwh/flr3 inversion-free genotype. In the mwh/TM3 genotype, in which mitotic crossing over is suppressed because of the inversion-heterozygosity, the observed spot frequencies were considerably reduced, but to different extents. In this genotype, spot induction by ellipticine was not statistically significant, and it was determined that >99% of the spots are due to mitotic recombination in mwh/flr3 flies. For the other compounds, spot induction in the inversion-heterozygous genotype was significant. The relative contribution of recombination to total spot induction in the inversion-free genotype was 88% for camptothecin. It was significantly lower for the two epipodophyllotoxins teniposide (71%) and etoposide (59%). Only suggestions can be proffered at present as to how these proportions could be related to the primary damage produced by the respective compounds on the chromosomes.