Alternative methods to evaluate the protective ability of sunscreen against photo-genotoxicity

Numerous epidemiological investigations show that sunlight is carcinogenic to humans and that the use of sunscreen may be effective in decreasing the risk of skin cancer. The biological activity of a sunscreen is evaluated by its ability to protect human skin from erythema as represented by a Sun Protection Factor (SPF). We propose that the sunscreen's protective effect against sunlight-induced genotoxicity, including mutation, should also be taken into account. In this study we examined the protective ability of sunscreens against natural sunlight and UV-induced genotoxicity in Drosophila somatic cells. We prepared three kinds of sunscreen samples, each with an SPF value of 20, 40 or 60 and compared their protective activities with commercial sunscreens. When a sunscreen of SPF 20, 40 or 60 was pasted on the plastic cover of a petri dish in which Drosophila larvae were exposed to the sun or UV lamps, genotoxicity decreased as the SPF of the sunscreen increased, relative to levels of genotoxicity observed in samples without sunscreen. However, the protective abilities of sunscreens were unexpectedly not so different from each other. To reveal the relationship between the protective activity of sunscreen and the wavelength of light with which larvae were irradiated through the sunscreen, we measured the transmittance of light through the petri dish cover on which the sunscreen was pasted. Effective protection was demonstrated by removing components of light whose wavelengths were below 315 nm. We suggest, that the measurement of anti-genotoxic activity and the determination of the wavelengths of light transmitted through the sunscreen should be an alternative method for evaluating the effectiveness of a sunscreen.</p

Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver

It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals

Chloroethylating anticancer drug-induced mutagenesis and its repair in Escherichia coli

Abstract Background Chloroethylnitrosourea (CENU) derivatives, such as nimustine (ACNU) and carmustine (BCNU), are employed in brain tumor chemotherapy due to their ability to cross the blood-brain barrier. They are thought to suppress tumor development through DNA chloroethylation, followed by the formation of interstrand cross-links (ICLs) that efficiently block replication and transcription. However, the alkylation of DNA and ICLs may trigger genotoxicity, leading to tumor formation as a side effect of the chemotherapeutic treatment. Although the involvement of O 6-alkylguanine-DNA alkyltransferase (AGT) in repairing chloroethylated guanine (O 6-chloroethylguanine) has been reported, the exact lesion responsible for the genotoxicity and the pathway responsible for repairing it remains unclear. Results We examined the mutations induced by ACNU and BCNU using a series of Escherichia coli strains, CC101 to CC111, in which reverse mutations due to each episome from F’101 to F’106 and frameshift mutations due to each episome from F’107 to F’111 could be detected. The mutant frequency increased in E. coli CC102, which can detect a GC to AT mutation. To determine the pathway responsible for repairing the CENU-induced lesions, we compared the frequency of mutations induced by CENU in the wild-type strain to those in the ada, ogt (AGT-deficient) strain, uvrA (nucleotide excision repair (NER)-deficient) strain, mismatch repair (MMR)-deficient strains, and recA (recombination deficient) strain of E. coli CC102. The frequencies of mutations induced by ACNU and BCNU increased in the ada, ogt strain, demonstrating that O 6-chloroethylguanines were formed, and that a portion was repaired by AGT. Mutation induced by ACNU in NER-deficient strain showed a similar profile to that in AGT-deficient strain, suggesting that an NER and AGT play at the similar efficacy to protect E. coli from mutation induced by ACNU. O 6-Chloroethylguanine is reported to form ICLs if it is not repaired. We examined the survival rates and the frequencies of mutations induced by ACNU and BCNU in the uvrA strain, the recA strain, as well as a double-deficient strain of CC102. The mutation profile of the double-deficient strain was similar to that of the NER-deficient strain, suggesting that an NER protects E. coli from mutations but not recombination. In addition, cell death was more pronounced in the uvrA, recA double-deficient strain than in the single-deficient strains. Conclusion These results suggest that the toxic lesions induced by CENU were repaired additively or synergistically by NER and recombination. In other words, lesions, such as ICLs, appear to be repaired by NER and recombination independently

The involvement of cell cycle checkpoint-mutations in the mutagenesis induced in Drosophila by a longer wavelength light band of solar UV

Solar ultraviolet radiation is considered to be injurious rather than necessary for most organisms living on the earth. It is reported that the risk of skin cancer in humans increased by the depletion of the ozone layer. We have examined the genotoxicity of solar ultraviolet, especially the longer wavelengths light, using Drosophila. Recently, we have demonstrated that light of wavelengths up to 340 nm is mutagenic on Drosophila larvae. Using an excision repair-deficient Drosophila strain (mus201), we have obtained results suggesting that the lesion caused in larvae by the 320 nm-light irradiation may be similar to the damage induced by irradiation at 310 nm, and that light of 330 and 340 nm may induced damage different from that induced by 310 and 320 nm-light. To examine the difference in DNA damage induced by light of particular wavelength, we performed monochromatic irradiation on larvae of two Drosophila strains; one excision repair-deficient (mei-9) and another postreplication repair-deficient (mei-41). 310 and 320 nm-light was more mutagenic in the mei-9 strain than mei-41, whereas 330 and 340 nm-light was more mutagenic in mei-41 than in mei-9. It is demonstrated that the mei-41 gene is a homologue of the human atm gene which is responsible for a cell cycle checkpoint. This result suggests that 310-320 nm-light induces DNA damage that is subject to nucleotide excision repair (NER) and that 3300-360 nm-light causes damage to be recognized by the cell cycle checkpoint but it is not repairable by NER

Increase of somatic cell mutations in oxidative damage-sensitive drosophila

Abstract Background Oxidative damage is an important genotoxic source for almost all organisms. To efficiently detect mutations induced by oxidative damage, we previously developed a urate-null Drosophila strain. Using this Drosophila strain, we showed the mutagenic activity of environmental cigarette smoke (ECS) and the herbicide paraquat, which are known to produce reactive oxygen species (ROS). In the present study, we examined the mutagenic activities of carcinogenic mutagens that are considered to cause mutations by adduct formation, alkylation, or crosslinking of cellular DNA in the oxidative damage-sensitive Drosophila to evaluate how the oxidative damage induced by these mutagens is involved in causing mutations. In addition, we evaluated whether these oxidative damage-sensitive flies may be useful for mutation assays. Methods We performed the wing-spot test in oxidative damage-sensitive Drosophila (urate-null strains) to examine the mutagenicity of 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), mitomycin C (MMC), 4-nitroquinoline N-oxide (4NQO), N-nitrosodimethyl-amine (NDMA), and N-nitrosodiethylamine (NDEA). We also observed the mutagenicity of X-ray irradiation as a control in which mutations should be mainly caused by oxidative damage. Results As expected, the mutagenic activity of X-ray irradiation was higher in the urate-null Drosophila than in the wild-type Drosophila. The mutagenic activities of the tested compounds were also higher in the urate-null Drosophila than in the wild-type Drosophila. In experiments using another urate-null strain, the mutagenicity of N-nitrosodialkylamines was also higher in the urate-null flies than in the wild-type ones. Conclusions The tested compounds in this study were more mutagenic in urate-null Drosophila than in wild-type Drosophila. It was supposed that ROS were generated and that the ROS might be involved in mutagenesis. The present results support the notion that in addition to causing DNA lesions via adduct formation, alkylation, or DNA crosslinking, these mutagens also cause mutations via ROS-induced DNA damage. As such, urate-null Drosophila appear to be useful for detecting the mutagenic activity of various mutagens, especially those that produce reactive oxygen. If the mutation rate increases on a mutation assay using urate-null Drosophila, it might suggest that the mutagen generates ROS, and that the produced ROS is involved in causing mutations

Exposure to Cigarette Smoke Increases Urate Level and Decreases Glutathione Level in Larval Drosophila melanogaster

Recently, we reported experimental evidence to support the notion that in Drosophila melanogaster, urate is involved in defense against toxic effects of environmental cigarette smoke (ECS). To obtain further information pertaining to the defense mechanisms involving urate and other antioxidants, the present study measured the levels of urate, its precursors and glutathione, and SOD activity in larval flies of wild-type strains (Oregon-R and Canton-S) and two urate-null mutant strains (ma-l and ry1) following exposure to ECS for various durations. In both wild type strains, unlike the case in either of the mutant strains, the urate level significantly increased above the basal level in a manner dependent on the duration of ECS exposure. Similar increases in the level of urate precursors were found in Canton-S and in both of the urate-null strains. There was a slight increase in glutathione level above the control level following ECS exposure for a short time, followed by an exposure-dependent decrease to less than 60% of the control level within the exposure range used in all of the four strains. On the other hand, no appreciable change was found in the SOD activity prior to or following ECS exposure, irrespective of the strain examined. In terms of the survival of treated larvae to adulthood under the conditions used for the measurements of urate and others, it was found that wild-type strain Canton-S was as sensitive as the urate-null mutant strains and clearly more sensitive than wild-type strains Oregon-R and Hikone-R. This was so despite the fact that, compared with Oregon-R, Canton-S contained urate at relatively higher levels prior to and following ECS exposure, and that the glutathione levels in Canton-S prior to and following treatment were comparable with those in other strains. These results are discussed with respect to the involvement of urate and glutathione in defense against the toxicity of ECS and the possible existence of another defense mechanism which is deficient in the Canton-S strain