In vivo biological activity of the components of haematoporphyrin derivative.

The in vivo biological activity of various fractions and components of haematoporphyrin derivative (HpD) have been determined by measuring the depth of necrosis of implanted tumours in mice exposed to light after the administration of standard doses of porphyrins dissolved in alkali. In this assay, haematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin are inactive, but the mono- and di-acetates of haematoporphyrin (which are major components of HpD) and acetoxyethylvinyldeuteroporphyrin are active. However, the situation appears to be more complex than this. The normal method for preparing HpD for injection involves an alkali treatment which causes hydrolysis and elimination of the acetoxy functions, and the only recognized products (haematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin) are inactive in the in vivo assay. It is concluded that the active component here is a porphyrin, possibly a dimer or oligomer, which is retained on the column during the normal separation by HPLC. This conclusion is supported by the observations that (i) the crude material obtained from the spent column is active without further alkali treatment, and (ii) activity develops over 30 min, when HpD or the mono- or diacetates of haematoporphyrin are treated with sodium bicarbonate in aqueous DMSO. The advantages of working with a pure substance (e.g. haematoporphyrin diacetate) rather than a mixture (HpD) are stressed

Prediction and assessment of the effects of mixtures of four xenoestrogens.

The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens

Comparative CYP-omic analysis between the DDT-susceptible and -resistant Drosophila melanogaster strains 91-C and 91-R

BACKGROUND: Cytochrome P450 monooxygenases (P450s) are involved in the biosynthesis of endogenous intracellular compounds and the metabolism of xenobiotics, including chemical insecticides.We investigated the structural and expression level variance across all P450 geneswith respect to the evolution of insecticide resistance under multigenerational dichlorodiphenyltrichloroethane (DDT) selection. RESULTS: RNA-sequencing (RNA-seq) and reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) indicated that the transcript levels of seven P450 genes were significantly up-regulated and three P450 genes were down-regulated in the DDT-resistant strain 91-R, as compared to the control strain 91-C. The overexpression of Cyp6g1 was associated with the presence of an Accord and an HMS-Beagle element insertion in the 5′ upstreamregion in conjunction with copy number variation in the 91-R strain, but not in the 91-C strain. A total of 122 (50.2%) fixed nonsynonymous (amino acid-changing) mutationswere found between 91-C and 91-R, and 20 (8.2%) resulted in amino acid changes within functional domains. Three P450 proteins were truncated as a result of premature stop codons and fixed between strains. CONCLUSION: Our results demonstrate that a combination of changes in P450 protein-coding regions and transcript levels are possibly associated with DDT resistance, and thereby suggest that selection for variant function may occur within this gene family in response to chronic DDT exposure

Genotoxic mixtures and dissimilar action: Concepts for prediction and assessment

This article has been made available through the Brunel Open Access Publishing Fund. This article is distributed under the terms of the creative commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s)and the source are credited.Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.UK Food Standards Agenc

Mixtures of four organochlorines enhance human breast cancer cell proliferation.

In view of the large differences between the concentrations of estrogenic chemicals needed to elicit effects in in vitro assays and their levels in human tissues, it is hard to explain possible health risks in terms of exposure to individual compounds. Human populations, however, are exposed to mixtures of estrogenic and estrogen-like agents and it is necessary to consider the impact of combined effects. We assessed the combined effects of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT), 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), beta-hexachlorocyclohexane (beta-HCH), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT) on the induction of cell proliferation in MCF-7 cells. All four compounds are persistent organochlorines that can be found in human tissues. We performed extensive concentration-response analyses with the single agents to predict the effects of two mixtures of all four compounds with different mixture ratios. We calculated the predictions by using the pharmacologically well-founded models of concentration addition and independent action and then tested them experimentally. o,p'-DDT, p,p'-DDE, beta-HCH, and p,p'-DDT acted together to produce proliferative effects in MCF-7 cells. The combined effect of the four agents could be predicted on the basis of data about single agent concentration-response relationships. Regression analysis demonstrated that there were combination effects even when each mixture component was present at levels at or below its individual no-observed-effect-concentration. We assessed combination effects in two ways: First, evaluations in relation to the proliferative responses induced by single mixture components revealed that the combination effects were stronger than the effects of the most potent constituent. Thus, according to this method of evaluation, the combined effects may be termed synergistic. Second, comparisons with the expected effects, as predicted by concentration addition and independent action, showed excellent agreement between prediction and observation. With this approach, the combined effect of all four compounds can be termed additive