The genetic toxicology of metal compounds: I. Induction of λ prophage in E coli WP2S(λ)

A number of metal compounds have been shown to be human carcinogens. Others, while not proven human carcinogens, are able to cause tumors in laboratory animals. Short-term bacterial assays for genotoxic effects have not been successful in predicting the carcinogenicity of metal compounds. We report here the ability of some metal compounds to cause the induction of λ prophage in E coli WP2_S(λ). By far the strongest inducing ability was observed with K2CrO4, followed by Pb(N0₃)₂ > MnCl₂ > Ni(OOCCH₃)₂ > CrCl₂ > NaWO₄ > Na₂MoO₄ > KMnO₄. With the exception of chromate, long-term exposures in a narrow, subtoxic dose range were required in order to demonstrate phage induction. A new microtiter assay for λ prophage induction, which incorporates these features, is described. This system also was able to detect very small amounts of organic carcinogens.     

XPA protein alters the specificity of ultraviolet light-induced mutagenesis in vitro.

Studies of ultraviolet (UV) light mutagenesis have demonstrated mutations at common sites in the target genes of shuttle vector plasmids replicated in cultured cells or by cellular extracts. The reasons for the specific pattern of mutagenesis are largely unknown. We have examined the specificity of UV-induced mutagenesis by replicating plasmid pLS189, irradiated with 40 J/m(2) UVC or unirradiated, in either xeroderma pigmentosum group A (XP-A) or HeLa cellular extracts. The XP-A extract displayed slightly lower replication ability, but produced a higher mutant frequency, compared to that of HeLa extract. Use of irradiated plasmid inhibited replication by an average of 63% and increased the mutant frequency by an average of 16.7-fold. Analysis of mutation spectra revealed nonrandom patterns of mutagenesis that differed significantly between HeLa and XP-A extracts. In comparison to HeLa extract, replication in XP-A extract resulted in lower frequencies of GC --> AT transitions and tandem double-base substitutions, and a higher frequency of deletions. Replication in HeLa extract produced hotspots at positions 100, 108, and 156 that were not produced by XP-A extract. Furthermore, XP-A extract produced hotspots at positions 124, 133, and 164, sites not characteristic of previous UV-induced mutagenesis studies using XPA-expressing cells. Addition of purified XPA protein to reactions containing XP-A extract altered each of these parameters, including loss of the hotspots at positions 124 and 133, to yield a more HeLa-like spectrum. These results indicate a previously uncharacterized role of the XPA protein in influencing the specificity of UV-induced mutagenesis during DNA replication.     

Characterization of Trp(+) reversions in Escherichia coli strain WP2uvrA

The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains. In contrast to Salmonella His(+) revertants, E.coli Trp(+) revertants have not been characterized at the molecular level. In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA). In spontaneous Trp(+) revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT). Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNA(Glu) (glnU), tRNA(Lys) (lysT) and tRNA(Tyr) (tyrT, tyrU), the Trp(+) reversion system with E.coli WP2uvrA detected five types of base substitutions, A.T-->T.A, A.T-->C.G, A.T-->G.C, G.C-->A.T and G.C-->T.A. About 30-50% of Trp(+) revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations. In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations. Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A.T base pairs (trpE65, lysT) and G.C base pairs (glnU, tyrT, tyrU).     

Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of interleukin-4 in ultraviolet B light-induced immunosuppression.

Prolonged exposure to ultraviolet light (UV) is known to lead to premature skin ageing, increased incidence of cataract and a high risk of developing skin cancers. UV-B irradiation, even if given as a single suberythemal dose, suppresses some immune responses, possibly reducing the production of T helper (Th) 1 cytokines [interleukin (IL)-2 and interferon-gamma] and augmenting Th2 cytokines (IL-4, IL-5 and IL-10) in mice. We investigated the role of IL-4 in UV-B induced immunomodulation using IL-4 knockout (IL-4 -/-) mice and the parent strain Bb129. Suberythemal UV-B irradiation (1440 J/m2) led to a reduction in the density and antigen presenting ability of Langerhans' cells in the epidermis of both normal and IL-4 -/- mice. Exposure also induced an accumulation of CD4+ and CD8+ lymphocytes as well as dendritic cells in the lymph nodes draining the irradiated site in both strains. The proliferation of lymph node cells in response to the mitogen concanavalin A was enhanced in the IL-4 -/- mice compared with the parent strain. Following UV-B exposure, this proliferation was increased in lymph node cells of parent mice but was significantly suppressed in the IL-4 -/- mice. The contact hypersensitivity (CH) response to oxazolone was suppressed to the same extent by UV-B irradiation in both strains. In the parent mice, infected with herpes simplex virus (HSV) following UV-B exposure and challenged subsequently with inactivated virus, the delayed hypersensitivity (DH) response was suppressed by about 50% compared with unirradiated mice; no such suppression in DH occurred in irradiated IL-4 -/- mice infected with HSV. Thus, IL-4 may be an important mediator of the UV-B-induced suppression in DH but not in CH, where other cytokines may be involved or may compensate for the lack of IL-4.     

RecA-independent mutagenesis in Escherichia coli: effects of umuC and mucB mutations

We have studied the effects of a umuC mutation on reversionof the lacZ19124 and lacZ19136 frameshift markers of Escherichiacoli. Introduction of a umuC:: Tn5 mutation into a strain carryingthe lacZ19136 marker resulted in enhanced reversion by 9-aminoacridineand the acridine half-mustard ICR191, whereas reversion of the^lacZ19124 marker was decreased (but not abolished) in a umuCstrain. The reversion frequency of the lazZ19136 marker wasdecreased by a derivative of pKM101 in which the ^mucB gene wasinactivated by a Tn5 insertion.The reversion frequency of thelazZ19124 mzrker was relatively unchanged by either plasmid.Since both 9-aminoacridine and ICR191 mutagenesis of these strainsis independent of the recA⁺ and lexA⁺ gene products, these resultsmay suggst a broader role for the UmuC protein in regulatinginduced mutation frequencies than has previously been suspected.The contrasting effects of the umuC and mucB mutations on reversionof the lacZ19136 marker may suggest a copy number effect, orperhaps more likely that are inherent (functional) differencesbetween the UmuC and MucB proteins. ²To whom correspondence should be addressed     

Enhancement of invasiveness of Yersinia enterocolitica and Escherichia coli in HEp-2 cells by centrifugation.

Centrifugation enhanced the infectivity of invasive Escherichia coli and Yersinia enterocolitica for HEp-2 cells. Noninvasive bacteria were not endocytosed after centrifugation. The centrifugation procedure may increase the sensitivity of testing for bacterial invasiveness in cell culture without causing false-positive results.     

Chemical mutagenesis testing in Drosophila. IX. Results of 50 coded compounds tested for the national toxicology program

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sex-linked recessive lethal (SLRL) assay. As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. One gaseous chemical (1,3-butadiene) was tested only by inhalation. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. These included bis(2-chloroethyl) ether, 1,4-butanediol diglycidyl ether, 1-chloro-2-propanol, dimethyl methylphosphonate, dimethyl morpholinophosphoramidate, dimethyloldihydroxy-ethylene urea, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isatin-5-sulfonic acid (Na salt), isopropyl glycidyl ether, and urethane. Five of these, including 1,4-butanediol diglycidyl ether, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isopropyl glycidyl ether, and urethane, also induced reciprocal translocations. © 1994 Wiley-Liss, Inc.     

Collaborative study of interlaboratory variability in Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101

A collaborative study of interlaboratory variability in bacterialmutagenicity induced by mitomycin C (MMC) and bleomycin (BLM)was performed using the four strains Salmonella typhimuriumTA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101.Thirteen laboratories participated in this study. The four strainsand two chemicals were sent from a central source to each laboratory.Each strain was cultured in nutrient broth containing antibioticsand laboratories were requested to ensure that the spontaneouscounts for marker check fell within a specified range in thepreparation of the original stock culture. Concerning the responseto the chemicals, most interlaboratory variability was withina 4-fold range for tests with TA102, TA2638 and WP2/pKM101.From the results of the statistical analysis using the linearregression test, positive results in TA102, TA2638 and WP2/pKM101and negative results in WP2 uvrA/pKM101 were obtained by MMCtesting in all of the laboratories. On the other hand, withBLM testing, considerable interlaboratory variability was observedbetween the strains, largely because of the variation in resultsof the repeat experiments. Overall mean spontaneous revertantcounts in all laboratories were within acceptable ranges forall four bacterial strains. In conclusion, it is judged thatamong Japanese laboratories, there is excellent agreement inthe tested response of these bacterial strains to a strong mutagensuch as MMC, as well as uniformity in the spontaneous reversionrates. However, for chemicals such as BLM that induce a weakresponse, it may be necessary to repeat experiments severaltimes to obtain clear results. In this study, TA102 was shownto be a useful strain for routine mutagenicity testing. Thisnecessitated control over culture maintenance by the additionof tetracycline and strict selection in the preparation of theoriginal stock cultures.     

Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis.

The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same.     

Impact of reactive oxygen species on spontaneous mutagenesis in Escherichia coli

Reactive oxygen species (ROS) are potent oxidants that attack chromosomal DNA and free nucleotides, leading to oxidative DNA damage that causes genetic alterations. To avoid the ROS-mediated mutagenesis, cells have elaborate mechanisms including powerful antioxidant components and repair pathways that eliminate oxidative DNA damage. Because of the effective anti-mutagenic functions, it has been unclear to what extent the ROS contribute to spontaneous mutagenesis. Here we show that a significant portion of spontaneous mutations is actually caused by the ROS in aerobically growing Escherichia coli cells. Using the rpsL gene as a mutational target sequence, we established an experimental procedure to analyze spontaneous mutations occurring under a strictly anaerobic condition. Strong mutator phenotypes of cells defective in both mutM and mutY genes or ones lacking mutT gene were completely suppressed under the anaerobic condition, indicative of an absence of hydroxyl radicals in the cells. From a series of analyses with wild-type E. coli cells grown under different redox conditions, it appeared that 89% of base substitutions were caused by the ROS, especially hydroxyl radicals, in cells growing in the atmosphere. The ROS-mediated spontaneous mutations included highly site-specific base substitutions, two types of randomly occurring transversions, G:C-->C:G and A:T-->T:A, and -1 frameshifts at non-iterated base sequences.     

Cobaltous chloride-induced mutagenesis in the supF tRNA gene of Escherichia coli

The spectrum of mutations induced by cobalt(II) chloride (CoCl2) was examined using plasmid pUB3 DNA, which was propagated after transfection into Escherichia coli SY1032/pKY241 host cells. The vector plasmid carried an E.coli supF suppressor tRNA gene as a target for mutations. After CoCl2 treatment, 64 independent nalidixic acid-resistant, ampicillin-resistant and Lac+ (SupF-) clones were obtained and the altered sequences of the mutated supF genes were determined. Deletions and frameshifts were the predominant mutational event (61%) induced by CoCl2 and base substitutions were induced to a lesser degree (29%). Analysis of sequence alterations at all the sites of mutation revealed that: (i) 18 of 19 base substitutions and eight of 10 frameshifts occurred at G:C sites, suggesting that the formation of N7G-Co(II) adducts may be responsible for premutagenic lesions of these mutations; (ii) short sequence repeats were mostly found at the sites of deletions and frameshifts. Slippage-misalignment is also suggested to be a mechanism for the induction of mutations at these sites.     

Regulation of the Shiga-like toxin II operon in Escherichia coli.

Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms. Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies. Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA). PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E. coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda. Both mechanisms are dependent on bacteriophage induction and hence are recA dependent. Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28. While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated.     

The genetic control of contact sensitization to inorganic metal compounds in guinea-pigs

Only a proportion of outbred guinea-pigs can be sensitized to K₂Cr₂O₇, BeF₂ and HgCl₂. Inbred Strain II can be sensitized to K₂Cr₂O₇ and BeF₂, but not to HgCl₂. Inbred strain XIII can be sensitized to HgCl₂ but not to K₂Cr₂O₇ or BeF₂. The ability to become sensitized appears to be transmitted as a dominant characteristic which is not sex linked.     

Some properties of purified Escherichia coli heat-stable enterotoxin II.

We examined the biological properties of purified Escherichia coli heat-stable enterotoxin II (STII) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT). The action of STII over time differed from those of STI and CT. STII did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells. Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT. This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT. Subsequently, we examined the involvement of prostaglandins in the action of STII. The level of prostaglandin E2 in the fluid accumulated as a result of the action of STII increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STII. These results implicate prostaglandin E2 in the mechanism of action of STII.     

Enhancement of Escherichia coli adherence to epithelial cells derived from estrogen-stimulated rats.

The effect of exogenous estrogen administered to male and oophorectomized female rats was investigated with regard to in vitro adherence of eight uropathogenic strains of Escherichia coli to exfoliated bladder and vaginal epithelial cells. Uroepithelial cells obtained from estrogenized male and estrogenized oophorectomized female rats and vaginal cells obtained from estrogenized oophorectomized female rats demonstrated significantly enhanced (P less than 0.005) host cell avidity for E. coli attachment, irrespective of bacterial adhesin expressed, when compared with such cells from nonestrogenized male and female oophorectomized rats. These animal studies suggest that female reproductive hormones may contribute to urinary-tract infection in premenopausal females by enhancing susceptibility to E. coli colonization of uroepithelial cells.