Lysyl-tRNA synthetase produces diadenosine tetraphosphate to curb STING-dependent inflammation.

Inflammation is an essential part of immunity against pathogens and tumors but can promote disease if not tightly regulated. Self and non-self-nucleic acids can trigger inflammation, through recognition by the cyclic GMP-AMP (cGAMP) synthetase (cGAS) and subsequent activation of the stimulator of interferon genes (STING) protein. Here, we show that RNA:DNA hybrids can be detected by cGAS and that the Lysyl-tRNA synthetase (LysRS) inhibits STING activation through two complementary mechanisms. First, LysRS interacts with RNA:DNA hybrids, delaying recognition by cGAS and impeding cGAMP production. Second, RNA:DNA hybrids stimulate LysRS-dependent production of diadenosine tetraphosphate (Ap4A) that in turn attenuates STING-dependent signaling. We propose a model whereby these mechanisms cooperate to buffer STING activation. Consequently, modulation of the LysRS-Ap4A axis in vitro or in vivo interferes with inflammatory responses. Thus, altogether, we establish LysRS and Ap4A as pharmacological targets to control STING signaling and treat inflammatory diseases.

Modulating the DNA Damage Response to Improve Treatment Response in Cervical Cancer.

Cervical cancer is the fourth most common cause of cancer-related death in women worldwide and new therapeutic approaches are needed to improve clinical outcomes for this group of patients. Current treatment protocols for locally advanced and metastatic disease consist of ionising radiation and chemotherapy. Chemoradiation induces cytotoxic levels of DNA double-strand breaks, which activates programmed cell death via the DNA damage response (DDR). Cervical cancers are unique given an almost exclusive association with human papillomavirus (HPV) infection; a potent manipulator of the DDR, with the potential to alter tumour sensitivity to DNA-damaging agents and influence treatment response. This review highlights the wide range of therapeutic strategies in development that have the potential to modulate DDR and sensitise cervical tumours to DNA-damaging agents in the context of HPV oncogenesis.

New horizons in the understanding of the causes and management of diabetic foot disease: report from the 2017 Diabetes UK Annual Professional Conference Symposium.

Diabetes-related foot disease remains a common problem. For wounds, classic teaching recommends the treatment of any infection, offloading the wound and ensuring a good blood supply, as well as ensuring that the other modifiable risk factors are addressed and optimized. There remain, however, several questions about these and other aspects of the care of diabetes-related foot disease. Some of these questions are addressed in the present report; in particular, the impact of newer technologies in the identification of any organisms present in a wound, as well as the use of novel approaches to treat infections. The use of new remote sensing technology to identify people at risk of developing foot ulceration is also considered, in an attempt to allow early intervention and prevention of foot ulcers. The psychological impact of foot disease is often overlooked, but with an increasing number of publications on the subject, the cause-and-effect role that psychology plays in foot disease, such as ulcers and Charcot neuroarthropathy, is considered. Finally, because of heterogeneity in diabetic foot studies, comparing results is difficult. A recently published document focusing on ensuring a standardized way of reporting foot disease trials is discussed.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR.

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.

AMP-kinase alpha2 subunit gene PRKAA2 variants are associated with total cholesterol, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol in normal women.

BACKGROUND: 5'-AMP-activated protein kinase (AMPK) inactivates critial ensymes in fatty acid and cholesterol synthesis. We hypothesised that the serum lipid profile may be influenced by genetic variation in the AMPK catalytic alpha2 subunit. METHOD: We examined association of 5 tagging SNPs (tSNPs) in the PRKAA2 gene with serum lipids in 2777 normal Caucasian females (mean age 47.4+/-12.5 years). RESULTS: All tSNPs were associated with total- and LDL-cholesterol, (p<0.001 to 0.034), explaining variances of 0.13-0.59% and 0.11-0.55% respectively. One haplotype (frequency 34.7%) showed lower total- and LDL-cholesterol compared with the most common haplotype (frequency 45.7%) (p< or =0.001), explaining 0.78% of total- and 0.75% of LDL-cholesterol. Another haplotype (frequency 10.5%) was significantly associated with lower HDL-cholesterol (p = 0.005), explaining 0.59% of variance. CONCLUSIONS: PRKAA2 gene variants are significantly associated with serum lipoproteins in a large sample of normal female Caucasians.

The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange.

Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity.

The Facility for Automated Experiments in Cell Biology (FAECB) collection of over 200 lines of ultraviolet (UV)-sensitive mutant Chinese hamster ovary (CHO) cells has previously been studied for complementation group assignment (CG), with representatives of rodent UV CGs 1-6 (ERCC1-6) and the new rodent XRCC9/FANCG group identified. Ten mutants from the collection, including a further six derived from wildtype AA8, three UV-sensitive double-mutants of CHO ERCC1 cell line UV4, and a UV-sensitive mutant of CHO XRCC1 cell line EM9, had not been assigned or characterized in these previous studies. These 10 mutants include 8 with approximately 1.5-fold the UV-sensitivity of the parental line (AA8, EM9, or UV4), and 2 with about 2-fold the UV-sensitivity of AA8. The present study reports the partial characterization of these 10 mutants in terms of sensitivity to UV (with and without caffeine), ionizing radiation, mitomycin C (MMC) and ethyl methanesulfonate (EMS); proficiency in DNA repair (unscheduled DNA synthesis (UDS)); and UV-mutability. The phenotypes of the 10 cell lines were heterogeneous, a number showed reduced UDS or UV-sensitization by caffeine, whilst others showed marked sensitivity to EMS or MMC, and they may have mutations in different genes involved in nucleotide excision repair, post-replicational repair, base excision repair or recombinational repair. Previous mutants isolated as part of the FAECB collection have proved to be extremely important in characterizing mammalian DNA repair processes and cloning human repair genes and these current mutants, whilst not as hypersensitive to UV, may still have the potential to make further contributions.

The XRCC2 human repair gene influences recombinational rearrangements leading to chromatid breaks.

PURPOSE: To test the possible involvement of the XRCC2 gene in the control of intra- versus interchromatid rearrangements leading to chromatid breaks in G2 cells by studying the colour-switch ratio (CSR) in harlequin-stained Chinese hamster irs1 cells. MATERIALS AND METHODS: The V79-4 mutant cell lines irs1 (XRCC2 mutation) and irs2 (XRCC8 mutation), two WT V79 lines and GT621-1 (irs1 transfected with the XRCC2 gene) were labelled with BrdU through two cell cycles, irradiated and sampled 1.5h after exposure. Metaphase spreads were analysed for chromatid break frequency and frequencies of colour-switch (colour-switch between chromatids at the break point) and non-colour-switch breaks, from which the CSR was calculated. RESULTS: Chromatid breaks were induced linearly with dose in all lines, and frequencies were elevated in irs1 and irs2 mutant cell lines when compared with WT lines. An XRCC2 transfected line (GT621-1) showed full radiosensitivity complementation with respect to frequencies of chromatid breaks. The CSR was significantly higher in irs1 (13.9%) than in the parental V79-4 (7.5%) or irs2 (4.9%) cells. GT621-1 cells showed partial, but significant complementation with respect to CSR (9.2%). CONCLUSIONS: It is concluded that the significantly higher CSR for the irs1 mutant than for the wild-type parental V79-4 line indicates the involvement of the XRCC2 gene product in the control of the rearrangement process leading to chromatid breaks.

Isolation of camptothecin-sensitive chinese hamster cell mutants: phenotypic heterogeneity within the ataxia telangiectasia-like XRCC8 (irs2) complementation group.

Using a replica microwell method, four Chinese hamster lines which exhibit hypersensitivity to the topoisomerase I inhibitor camptothecin, designated CM1, CM2, CM3 and CM6, have been isolated. Their sensitivity towards camptothecin varied from 3.5- to 8.2-fold with relative sensitivity as follows: CM2 < CM3 < CM6 < CM1. Genetic analysis of the CM mutants has established that CM1, CM3 and CM6 fail to complement each other and can each be assigned to the irs2 (XRCC8) complementation group. The mutant CM2 could not be definitively assigned to a complementation group because it presented a semi-dominant phenotype. In contrast to their sensitivity to camptothecin, the four CM mutants were less sensitive (1.1- to 2.2-fold) to the topoisomerase II inhibitors etoposide and adriamycin, although CM1, CM3 and CM6 were more sensitive (2.5- to 3. 8-fold) to streptonigrin (a free radical generator and a topoisomerase II inhibitor). All four mutant lines displayed an increased sensitivity to the bifunctional alkylating agent mitomycin C (2.4- to 5.1-fold). Surprisingly, given their assignment to the irs2 (XRCC8) complementation group, CM1, CM3 and CM6 displayed only a minor increase in sensitivity to ionizing radiation (1.6-fold or less). Similar sensitivity of these CM mutants was observed for the radiomimetic compound bleomycin (1.7-fold sensitive or less). This study indicates that XRCC8 mutants are isolated at high frequency from the parent line V79 and that phenotypic heterogeneity amongst the irs2 (XRCC8) complementation group is greater than previously encountered. Mutations in different regions of the XRCC8 gene may be responsible for the differing cellular phenotypes. Hamster XRCC8 mutants show phenotypic similarities to cultured cells from ataxia telangiectasia and Nijmegen break syndrome (NBS) patients and are likely to be defective in the same pathway in which the ATM (ataxia telangiectasia-mutated) and the NBS genes operate.

Biomonitoring the human population exposed to pollution from the oil fires in Kuwait: analysis of placental tissue using (32)P-postlabeling.

The placenta is a readily available source of material for molecular epidemiological investigations. As such, DNA damage in this tissue can be indicative of maternal exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs). Previous reports have demonstrated that (32)P-postlabeling (PPL) is able to detect the presence of aromatic adducts in human placenta that are associated with maternal smoking during pregnancy. Using PPL we have assayed the DNA damage in placental samples from Kuwaiti mothers who were exposed to environmental pollution during pregnancy. This pollution arose in the aftermath of the Iraqi invasion of Kuwait, which left hundreds of oil wells burning. For comparison, further Kuwaiti samples were obtained approximately 1 year after the oil well fires and, as such, are from individuals unexposed to the airborne pollution from the oil well fires during pregnancy. In addition, placental samples were obtained from subjects in the United Kingdom. Adduct levels were measured in all samples using both the nuclease P1 and butanol extraction enhancement procedures. No elevation of adduct levels was observed in the placenta of mothers exposed to the oil well fires (n = 40) with either procedure (144 +/- 30 attomol/microg DNA for nuclease P1 enrichment, 245 +/- 50 attomol/microg DNA for butanol extraction), when compared with the nonexposed Kuwaiti mothers (180 +/- 32 and 281 +/- 39 attomol/microg DNA, respectively, n = 24). Similar adduct levels were observed in UK mothers who smoked cigarettes (178 +/- 30 and 284 +/- 52 attomol/microg DNA, n = 30), which in turn were approximately twice those observed in nonsmoking mothers (90 +/- 14 and 141 +/- 15 attomol/microg DNA, n = 12), although there is no significant difference in the distribution of adduct levels when statistical analysis is performed. Comprehensive interpretation of the Kuwaiti data is difficult as precise information on PAH levels is unavailable, although the data do seem to indicate that exposure to PAHs was not biologically significant.

The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants.

Using a replica plating microwell method, three Chinese hamster V79-derived cell lines, designated ETO1, ETO2 and ETO3, which exhibit hypersensitivity to the non-intercalating topoisomerase II inhibitor etoposide have been isolated. Mutant lines ETO2 and ETO3 are cross-sensitive to the topoisomerase II inhibitors adriamycin and streptonigrin; however, neither mutant is sensitive to the topoisomerase I inhibitor camptothecin, the bifunctional alkylating agent mitomycin C, nor hydrogen peroxide. In contrast, ETO1 is cross-sensitive to camptothecin but displays only slight sensitivity to adriamycin, streptonigrin and hydrogen peroxide, and is not sensitive to mitomycin C. It has been established through extensive cell fusion studies that all three mutants are genetically distinct, and that ETO2 and ETO3 genetically complement all other known etoposide-sensitive Chinese hamster cell mutants (i.e., irs1, XR-1, xrs1, V3, BLM2, ADR1, ADR3, ADR4 and ADR5) thus defining two new complementation groups of etoposide sensitive mutants. Interestingly, the hybrids created by the fusion irs2TOR (thioguanine and ouabain resistant)xETO1 and the reciprocal cross ETO1TORxirs2 both exhibited a response to camptothecin intermediate with respect to V79 and ETO1. It has been hypothesised that this partial complementation may be the result of intragenic complementation and that both ETO1 and irs2 result from mutations in the gene XRCC8. This study indicates that cellular responses to topoisomerase II inhibitors are complex and hypersensitivity may result from mutations in many different genes.

Mechanisms of genome maintenance and rearrangement: current research and recent advances in DNA repair and recombination.

The topics of the talks at the annual DNA Repair Network Meeting at City University, London were as usual wide-ranging and provided an absorbing programme. Covered in the 17 talks were the autoproteolysis of O(6)-methylguanine DNA alkyltransferase in Escherichia coli; identification of new intermediates in meiotic recombination in Saccharomyces cerevisiae; the SMC (structural maintenance of chromosomes) family of proteins in Schizosaccharomyces pombe; transposition and V(D)J recombination; mammalian Rad51 foci formation in Rad54, Rad52, XRCC2 and XRCC3 mutants; biochemical analysis of DNA-PK, ATM (ataxia telangiectasia mutated) and ATR (AT related); other human DNA repair deficiencies and their incidence, including xeroderma pigmentosum and a new DNA ligase IV-deficient patient, and back, once again, to alkyltransferase, this time in humans and its manipulation for engineering drug resistance in bone marrow for cancer treatment.

Recent advances in DNA repair and recombination.

The subjects of the talks at this 1-day DNA Repair Network meeting, held at City University, London on December 15, 1997, encompassed a range of topics and reflected some of the current areas of research in the United Kingdom. Topics included DNA double-strand break repair, V(D)J recombination, DNA ligases, the RecQ family of helicases and Bloom's syndrome, UVB and immunosuppression, the repair of oxidative damage and mismatch repair mechanisms.

XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages.

The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

Report on the meeting held at City University, London on 16 December 1996: recent progress in DNA repair, mutation and recombination.

This one-day DNA repair meeting was held at City University, London for the fourth consecutive year (P. Strike, Recent advances in DNA repair, mutation and recombination-a report of the meeting of the British Photobiology Society, London, 20 December 1993, Mutation Res. 315 (1994) 75-84; P. Strike, Recent advances in DNA repair and recombination: a report of the DNA Repair Network held at City University, London on 19 December 1994, Mutation Res. 337 (1995) 61-71 ; N.J. Jones, P. Strike, Recent research in DNA repair, mutation and recombination: a report of the DNA Repair Network meeting held at City University, London on 18 December 1995, Mutation Res. 364 (1996) 13-23). The programme consisted of 18 pre-offered talks, there being no specially invited speakers. The meeting was well attended with over 150 participants representing 23 universities, research institutes and hospitals the length and breadth of the United Kingdom. Overseas attendees represented laboratories from Japan, The Netherlands and Spain. The topics of the talks were varied and included mismatch repair, the repair of oxidative damage, transcription-coupled repair and various mutagen-hypersensitive human syndromes. Although wide-ranging, one recurring themes in many of the talks was that of homologous proteins and the correspondence of repair mechanisms in bacterial, fungal and mammalian systems.

Comparative induction of micronuclei in repair-deficient and -proficient Chinese hamster cell lines following clastogen or aneugen exposures.

The Chinese hamster cell line V79-4 and two ionising radiation-sensitive mutants irs1 and irs3, were used to assess the genotoxic effects of a clastogen (X-rays), an aneugen (podophyllotoxin) and an agent having both activities (etoposide), using the cytokinesis-block micronucleus assay combined with kinetochore labelling. All three agents induced micronuclei in a dose-dependent manner. Between 70 and 90% of the micronuclei induced by X-rays were kinetochore-negative and indicative of clastogenic activity, the relative frequencies of which were increased in the repair-deficient cell lines. In contrast, podophyllotoxin induced mainly kinetochore-positive micronuclei (up to 97%) and could be classified as an inducer of chromosome lagging and was essentially aneugenic in its activity. Micronuclei induced by etoposide were 61-84% kinetochore-negative and 26-39% kinetochore-positive, indicating both clastogenic and aneugenic activity. In the case of both etoposide and podophyllotoxin, there were no observed differences in the induction of micronuclei between the wild-type and repair-deficient cell lines. The data demonstrate the effectiveness of the cytokinesis-block micronucleus/kinetochore assay in determining the mechanisms of action of both chemicals and radiations upon chromosome structure and number and the potential influences of repair upon micronuclei induction.

Recent research in DNA repair, mutation and recombination: a report of the DNA Repair Network meeting, held at City University, London on 18 December 1995.

The now traditional one day Christmas DNA Repair meeting was held at City University, London for the third year in succession. With over 130 participants and a programme consisting of a total of 24 pre-offered presentations the meeting reached record dimensions. Attendees were from 24 institutions throughout the United Kingdom, and with several distinct research groups contained within the large contingents from the ICRF Clare Hall Laboratories and the MRC Cell Mutation Unit in Brighton, this indicates the increasing interest and depth of UK research in DNA repair. One slight disappointment of the meeting was the fall in the numbers of non-UK participants. Although the meeting in 1994 (Strike, 1995) saw an increase in presentations from Continental Europe (six countries including France, Germany. The Netherlands and Switzerland), the trend did not continue this year, with only Denmark being represented. The 24 contributors consisted of approximately equal numbers of postgraduate students, postdoctoral researchers and more "established' scientists reflecting the continuing policy of encouraging younger members of the repair community to present their work. The mix of presenters was particularly well illustrated by two excellent and consecutive talks by Professor Bryn Bridges (MRC Cell Mutation Unit) and Alison Mitchell, a postgraduate student in Stephen West's laboratory (ICRF, Clare Hall). The organisms under study were as equally disparate and included Archaebacteria, Escherichia coli. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus, mice and men. The range of topics was also varied and included bacterial mutagenesis, NMR studies of Ada protein, preferential DNA repair, cell cycle checkpoint genes, reconstitution of nucleotide excision repair and V(D)J recombination in vitro, creation of repair deficient transgenic mice and mismatch defects in human cells. The result was a very successful meeting which was characterized by the consistently high quality of both talks and posters.

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity.

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

Biomonitoring human exposure to environmental carcinogenic chemicals.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.