Escherichia coli responses to a single DNA adduct.

To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells.

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.

Cellular response to exocyclic DNA adducts.

The mutagenic potential of three exocyclic DNA adducts was studied in Escherichia coli and simian kidney cells by incorporating them into single-stranded DNA. Differences in the mutagenic potency of the adducts were observed between hosts: 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine were more mutagenic in simian cells, whereas 1,N2-(1,3-propan-1,3-diyl)-2'-deoxyguanosine was more mutagenic in E. coli. To investigate the cellular response to DNA adducts, a double-stranded DNA vector system was developed. Use of this system showed that 1,N6-ethenodeoxyadenosine blocks DNA synthesis strongly, and DNA synthesis past this adduct was highly accurate in E. coli. The blockage of DNA synthesis was overcome in an error-free manner by the recombination repair mechanism (daughter-strand gap repair).

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation.

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.

A selective difference between human Y-chromosomal DNA haplotypes.

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] [11] and as many as 5% of males are infertile [11] [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.

1,N6-ethenodeoxyadenosine, a DNA adduct highly mutagenic in mammalian cells.

1,N6-Ethenodeoxyadenosine (epsilon dA) is one of four exocyclic DNA adducts produced by chloroethylene oxide and chloroacetaldehyde, reactive metabolites of vinyl chloride, a human carcinogen. epsilon dA has also been detected in DNA of the liver of humans and untreated animals, suggesting its formation from endogenous sources. The mutagenic potential of epsilon dA was studied using a single-stranded shuttle vector system in several E. coli strains and in simian kidney cells (COS7). This vector system enables quantitative analysis of translesional synthesis past a site-specifically placed DNA adduct in both hosts owing to the lack of the complementary strand. In experiments with five strains of E. coli, a very limited number of targeted mutations (one epsilon dA-->T, one epsilon dA-->dC, and two epsilon dA-->single base deletion) were observed among 756 transformants in hosts preirradiated with UV; no targeted mutations were observed among 563 transformants in nonirradiated hosts. These results indicate that nonmutagenic base pairings of epsilon dA:T are the almost exclusive events in E. coli. In COS7 cells, the frequency of targeted mutations was 70%, consisting of epsilon dA-->dG (63%), epsilon dA-->T (6%), and epsilon dA-->dC (1%), indicating that the insertion of dCMP opposite the adduct is predominant. When compared with the results for 3,N4-ethenodeoxycytidine (epsilon dC), which was studied previously in the same system [Moriya et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11899-11903], the results of this study indicate that the intrinsic mutagenic potency of epsilon dA is comparable to that of epsilon dC in mammalian cells.

UVM, an ultraviolet-inducible RecA-independent mutagenic phenomenon in Escherichia coli.

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or inaccessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior UV irradiation of delta recA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVM (UV modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase III, we have examined mutagenesis at an epsilon C residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in UV-irradiated recA+, and in delta recA cells. These data indicate that UV irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap-filling DNA synthesis as well as during normal DNA replication.

Perspectives of recycling gamma irradiated sewage-sludge in agricultural applications: a study on methi (Trigonella foenum-graecum L.: leguminosae).

The effects of gamma-irradiated sludge on the growth and yield of methi (Trigonella foenum-graecum L.) in pot cultures have been studied. Gamma-irradiated sludge was found to inhibit the shoot length after 45 and 90 days of plant growth compared to plants grown in soil containing unirradiated sludge. The untreated sludge did not exhibit any detrimental effect on the shoot length of plants compared to the control. The root length of plants grown in soil supplemented with either gamma-irradiated or unirradiated sludge was found to be inhibited after 45 days of growth. However, irradiation of sludge resulted in the higher inhibition of root length of plants compared to when unirradiated sludge was used. The gamma-irradiated sludge appeared to negatively affect the physical growth parameters of the plant. The significant positive effect of gamma-irradiated sludge was observed on the biochemical growth parameters and yield of methi plants. There was a 3.5-, 1.7- and 2-fold increase in the total protein content, total soluble sugars and starch content, respectively, of plants grown in soil supplemented with gamma-irradiated sludge after 45 days of growth. The gamma-irradiated sludge did not show any detrimental effect on any of the three biochemical parameters studied, even after 90 days of plant growth. The sludge obtained from the conventional treatment process was found to be inhibitory to the protein and starch content of plants in the latter stages of plant growth. A significant increase in the yield of methi plants, after 45 as well as 90 days, grown in the presence of gamma-irradiated sludge indicates a beneficial effect of recycling of irradiated sludge for agricultural applications.

Potential of recycling gamma-irradiated sewage sludge for use as a fertilizer: a study on chickpea (Cicer arietinum).

The effects of gamma-irradiated sludge on the growth and yield of chickpea (Cicer arietinum) in pot cultures have been studied. Compared to plants grown only in soil, root length, fresh weight and dry weight of plants grown in soil supplemented with unirradiated sludge were found to be significantly reduced. This inhibition in growth was found to be nullified when plants were grown in soil supplemented with gamma-irradiated sludge, suggesting that gamma radiation induced inactivation of toxic substance(s) in sludge. The protein content of plants grown in soil supplemented with irradiated sludge was also found to be significantly increased compared to those grown with unirradiated or no sludge, after 45 days. There was no significant effect of gamma irradiated sludge on shoot length, total soluble sugars, starch content and yield of chickpea plants. The results obtained suggest that the sludge tested, and obtained from the digester of a conventional domestic sewage treatment plant, is inhibitory to several growth parameters. Gamma irradiation of sewage resulted in removal of this inhibition. This suggests a possibility of beneficial and safe recycling of gamma-irradiated sludge for agricultural uses.

Effect of gamma-irradiated sludge on the growth and yield of rice (Oryza sativa L. var. GR-3).

The effects of gamma-irradiated sludge on the growth and yield of rice (Oryza sativa L. var. GR-3) in pot cultures have been studied. Compared to plants grown only in soil, shoot length, root length, fresh weight, dry weight, total proteins, total soluble sugars, starch and chlorophyll content of plants grown in soil supplemented with unirradiated or gamma-irradiated sludge were found to be significantly increased. Irradiation of sludge significantly stimulated the linear growth of shoot and root systems as well as fresh and dry weights of plants, compared to those grown in soil containing unirradiated sludge. There was also an improvement in the grain yield (weight of seed) when plants were grown in soil supplemented with irradiated sludge. The results obtained suggest that the gamma-irradiated sewage sludge can be beneficially recycled for agricultural uses.

Inactivation of bacteria in sewage sludge by gamma radiation.

The survival of certain bacterial cultures suspended in sewage sludge and exposed to gamma-radiation was studied. The inactivation patterns of most of the organisms were significantly different when irradiation was performed using sewage samples collected in the summer and monsoon seasons. The summer sample collected from the anaerobic digestor afforded significant protection to both Gram negative and Gram positive organisms. This was evident by the increase in dose required to bring about a 6 log cycle reduction in viable count of the bacterial cultures, when suspended in sewage samples instead of phosphate buffer. The observations made using monsoon digestor samples were quite different. This sewage sludge greatly enhanced inactivation by gamma-radiation in most cases. The effects of certain chemicals on the inactivation patterns of two organisms-Salmonella typhi and Shigella flexneri-were examined. Arsenate, mercury and lead salts sensitised S. typhi, while barium acetate and sodium sulphide protected this culture against gamma-radiation. In the case of Sh. flexneri, barium acetate and iodacetamide proved to be radioprotectors. The effects of some chemicals on the inactivation pattern of Sh. flexneri cells irradiated in sludge are also discussed.