Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation assays

A novel technique for the selection of mutated DNA sequences,termed mismatch cleavage-polymerase chain reaction (MC-PCR),is proposed. The method is based on hybridizing genomic DNAwith a suitable probe, several 100 bp long. Mutated DNA sequenceswill form mismatched heteroduplexes which are cleaved by usingresolvases. Cleaved heteroduplexes are detected by ligationto an oligonucleotide adaptor and then amplified by using PCR.If practical, this technique would have considerable advantagesover the restriction site mutation (RSM) method. Failure toachieve cleavage efficiencies of close to 100% will not compromisesuccess. This is because positive signals (PCR amplification)arise from cleaved mutated sites and not, as in RSM, from DNAsequences resistant to cleavage by restriction endonucleases.Furthermore, the mutational target is much larger than in RSM.It would be possible to screen stretches of DNA several 100bp in length for mutations. Any mutation, independent of itslocation, could be identified. The usefulness of MC-PCR forthe genotypic selection of mutants will depend on the effectivenesswith which a small number of mismatched heteroduplexes can berecognized, cleaved and ligated. ¹To whom correspondence should be addressed     

Breast cancer, oestrogens and environmental pollutants: a re-evaluation from a mixture perspective

The continuing rise in breast cancer incidence rates in almost all Western countries cannot be explained entirely in terms of known risk factors of the disease process. Additional determinants need to be examined, among them exposure to environmental pollutants. With the demonstration that elevated levels of endogenous sex hormones and the use of hormones for the relief of menopausal symptoms are associated with the disease, the oestrogen hypothesis of breast cancer has received further empirical support. This has led to heightened concerns about the possible involvement of oestrogen-like pollutants, such as p,p'-Dichlorodiphenyldichloroethane (DDE). Case-control studies assessing whether women with breast cancer have elevated burdens of p,p'-DDE have produced results not suggestive of a link with cancer risks. However, possible effects of p,p'-DDE and other pollutants cannot be seen in isolation from sex hormones also present in serum. In view of the low potency of p,p'-DDE, and its low levels in serum, it seems unlikely that the chemical on its own will add significantly to the action of sex hormones. However, chemical analyses show that numerous pollutants with oestrogen-like effect profiles can be found in human tissues. Thus, the oestrogen hypothesis of breast cancer should be extended to encompass the multitude of these agents. Viewed from such a perspective, a putative role of organochlorines and other agents in breast cancer should not be dismissed prematurely. Instead, breast cancer epidemiology should face the reality of combined exposures and should take account of recent evidence from in vitro models demonstrating that a large number of oestrogen-like pollutants, all present at low levels, can act together to add to the internal oestrogenic load.     

The reduction of chromate is a prerequisite of chromium binding to cell nuclei

Isolated calf thymus nuclei bound a chromium(III) glutathione complex in a time-dependent manner. In contrast chromium(VI) (sodium chromate) did not bind. However, when chromate was incubated with the nuclei in the presence of glutathione, chromium adducts were detected. These observations indicate that the reduction of chromate, by a reducing agent such as glutathione, is a prerequisite for the generation of bonds between the metal and constituents of the cell nuclei in vitro. Chromium adducts with nuclei are probably one cause of DNA lesions and mutations.     

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.     

The formation of DNA cleaving species during the reduction of chromate by ascorbate

A detailed study of the ability of chromate in combination withascorbate to induce DNA single-strand breaks in the absenceof iron(II) and copper(II) has been carried out. In solutionscontaining 1 mM ascorbate and chromate in the range 0.1–1mM extensive DNA cleavage occurred. Chromate alone or the finalproduct of the chromate/ascorbate reaction were not responsiblefor the cleavage observed. Evidence is presented that an intermediategenerated during the reduction of chromate is the reactive species.No strand breaks occurred upon addition of catalase, pointingto a role for peroxidic species in the steps leading to thegeneration of the cleaving species. The exclusion of oxygenled to a substantial decrease in the number of strand breaks.Furthermore, the formation of strand breaks declined with decreasingconcentrations of phosphate in the phosphate buffers used asthe incubation medium. No DNA strand breaks were induced inmedium containing HEPES. These observations rule out chromium(V)as the agent directly responsible for the DNA degradation, aschromium(V) is formed during the reduction of chromate by ascorbatein HEPES buffer. Our results lead us to suggest that the DNA-damagingability of chromate upon reduction by ascorbate arises fromthe activation of oxygen exacerbated by phosphate and pointsto a peroxo or superoxo complex involving chromium(V) or chromium(IV)as a possible candidate.     

Studies of the binding of chromium(III) complexes to phosphate groups of adenosine triphosphate

The binding of a number of chromium complexes to ATP has been studied by 31P NMR spectroscopy. The results are consistent with the formation of outer-sphere complexes between ATP and the chromium species. The magnitude of the binding constant has been determined as approximately 500 mol-1 dm3 (pH = 7.00) for the interaction of trisethylenediamine chromium(III) with ATP. Under our experimental conditions no NMR signals are observed from complexes in which chromium(III) is bound directly to the phosphate groups of ATP. The relative stability of the inner sphere complexes of chromium(III) and glutathione or ATP is probably not a significant factor in determining the toxicity of chromium(III) complexes.     

Dysgenesis and Histological Changes of Genitals and Perturbations of Gene Expression in Male Rats after In Utero Exposure to Antiandrogen Mixtures

We investigated the ability of a mixture of three androgen receptorantagonists to induce disruption of male sexual differentiationafter perinatal exposure. The aim was to assess whether thejoint effects of vinclozolin, flutamide, and procymidone canbe predicted based on dose-response data of the individual chemicals.Chemicals were administered orally to pregnant Wistar rats fromgestational day 7 to postnatal day 16. Changes in reproductiveorgan weights and of androgen-regulated gene expression in prostatesfrom male rat pups were chosen as end points for extensive dose-responsestudies. With all end points, the joint effects of the threeantiandrogens were dose additive. Histological evaluations showedthat dysgenesis and hypoplasia of prostates, seminal vesicles,and epididymis were seen with the highest mixture doses. Nochanges were observed in any single-compound low-dose groupfor these lesions, nor were there histopathological changesin the testes. Pronounced dysgenesis of external genitals wasobserved with all doses of the mixture, and severe dysgenesiswas seen with a mixture for which the individual compounds causedno effects. A combination of doses of each chemical that onits own did not produce significant reductions in the weightsof seminal vesicles and PBP C3 expression induced a marked mixtureeffect. Thus, antiandrogens cause additive effects on end pointsof various molecular complexities such as alterations at themorphological and the molecular level. Exposure to antiandrogens,which appears to exert only small effects when judged on a chemical-by-chemicalbasis, may induce marked responses in concert with, possiblyunrecognized, similarly acting chemicals.     

The interaction potential of herbal medicinal products: a luminescence-based screening platform assessing effects on cytochrome P450 and its use with devil's claw (Harpagophyti radix) preparations

Objectives Potential interactions between herbal medicinal products and the cytochrome (CYP) P450 system are an important safety concern. We set out to develop a screening panel for assessing such interactions and use it to evaluate the interaction potential of devil's claw. Methods The panel consisted of luminescence‐based inhibition assays for CYP1A2, 2C9, 2C19, 2D6 and 3A4, and a reporter gene (luciferase) assay for pregnane X receptor (PXR) activation and CYP3A4 induction. Caftaric acid and chlorogenic acid, two compounds with strong fluorescence quenching properties, were used to demonstrate the assay's resistance to interference. We tested 10 commercial devil's claw preparations as well as harpagoside and harpagide, two important constituents of devil's claw. Key findings Five preparations were found to weakly inhibit CYP3A4 (IC50 124.2–327.6 µg/ml) and five were found to weakly activate PXR (EC50 10.21–169.3 µg/ml). Harpagoside and harpagide did not inhibit CYP3A4. In agreement with published data, bergamottin, a natural product known to interact with CYP3A4, was shown to inhibit CYP3A4 with an IC50 of 13.63 µm and activate PXR with an EC50 of 6.7 µm . Conclusions Devil's claw preparations are unlikely to have a clinically relevant effect on CYP function. The assay panel proved effective in screening devil's claw preparations, demonstrating its suitability for use with plant extracts. It showed superior sensitivity and resistance to interference.     

The suitability of concentration addition for predicting the effects of multi-component mixtures of up to 17 anti-androgens with varied structural features in an in vitro AR antagonist assay

The risks associated with human exposures to chemicals capable of antagonising the effects of endogenous androgens have attracted considerable recent interest. Exposure is typically to large numbers of chemicals with androgen receptor (AR) antagonist activity, yet there is limited evidence of the combined effects of multi-component mixtures of these chemicals. A few in vitro studies with mixtures of up to six AR antagonists suggest that the concept of concentration addition (CA) provides good approximations of experimentally observed mixture effects, but studies with larger numbers of anti-androgens, and with more varied structural features, are missing. Here we show that the mixture effects of up to 17 AR antagonists, comprising compounds as diverse as UV-filter substances, parabens, perfluorinated compounds, bisphenol-A, benzo(α)pyrene, synthetic musks, antioxidants and polybrominated biphenyls, can be predicted well on the basis of the anti-androgenicity of the single components using the concept of CA. We tested these mixtures in an in vitro AR-dependent luciferase reporter gene assay, based on MDA-kb2 cells. The effects of further mixtures, composed of four and six anti-androgens, could be predicted accurately by CA. However, there was a shortfall from expected additivity with a ten-component mixture at two different mixture ratios, but attempts to attribute these deviations to differential expression of hormone-metabolising CYP isoforms did not produce conclusive results. CA provides good approximations of in vitro mixture effects of anti-androgens with varying structural features.