Genotoxicity of cocoa in a series of short-term assays

Cocoa powder was evaluated for genotoxic activity and found to be inactive in the Ames assay, the mouse lymphoma assay, cytogenetic assays measuring chromosome breakage and SCE, and a cell transformation assay using Balb/c-3T3 cells. Although pure theobromine has been shown to be active in some of these test procedures, the levels of this methylxanthine present in cocoa powder were insufficient to elicit responses in this battery of tests.     

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells

Nitroarenes are ubiquitous environmental pollutants displaying potent genotoxicity in bacterial and mammalian cells. In this study, 2,4,7-trinitro-9-fluorenone (TNF) was more potent than 1-nitropyrene (1-NP) in eliciting genotoxic responses in 4 mammalian cell lines. All 4 cell types were capable of activating the nitroarenes, since no special incubation conditions were required. Inhibition of normal DNA synthesis and cytotoxicity were significantly increased with TNF in a dose range of 0.2-5 micrograms/ml for human teratocarcinoma (PA1) cells, mouse Sertoli (TM4) cells, rat hepatoma (RL12) cells, and human-Chinese hamster ovary (CHO-K1) cells. For 1-NP, a dose range of 10-20 micrograms/ml was required to achieve comparable results for the respective cell lines. Only the RL12 and CHO-K1 cells showed recovery of normal DNA synthesis when TNF or 1-NP was removed from the medium. The other cell types showed little or no recovery up to 42 h after removal of the nitroarene. In exclusively studying TNF, the induction of sister-chromatid exchanges (SCEs) and a delay in cell cycle as monitored by harlequin chromosomes, were observed at a concentration range of 0.003-0.2 microgram/ml in PA1, TM4, and RL12 cells. In CHO-K1 cells, TNF at 0.001-1 microgram/ml was clearly mutagenic at the hprt locus.     

Evasion of mammalian defense systems by orthopoxviruses

In the course of evolution, viruses have mastered various molecular mechanisms to evade defense reactions of the host organism. The understanding of these mechanisms would promote better comprehension of the crucial reactions directed against infectious agents and further insights into their organization and functioning. A considerable contribution to this field of study can be made by investigating orthopoxviruses pathogenic for humans, such as variola, monkeypox, cowpox, and vaccinia viruses. The experimental data reviewed here suggest that variola virus and other orthopoxviruses, in comparison to other virus families, possess an unsurpassed set of genes whose protein products efficiently modulate the diverse defense reactions of the host.     

Evasion of mammalian organism defense systems by orthopoxviruses

Viruses during their evolution have mastered various molecular mechanisms to evade the defense reactions of the host organism. When understanding the mechanisms used by viruses to overcome manifold defense systems of the animal organism, represented by molecular factors and cells of the immune system, we would not only comprehend better, but also discover new patterns of organization and function of these most important reactions directed against infectious agents. Here, study of the orthopoxviruses pathogenic for humans, such as variola, monkeypox, cowpox, and vaccinia viruses, may be most important. Analysis of the experimental data, carried out in this review, allows to infer that variola virus and other orthopoxviruses possess an unexampled set of genes whose protein products efficiently modulate the manifold defense mechanisms of the host organisms compared with the viruses from other families.     

Biotransformation of drugs by microbial cultures for predicting mammalian drug metabolism

This review discusses the microbial transformation studies of drugs, correlating them with the corresponding metabolism (biotransformation) in animal systems. Approaches are provided for development of microbial models for mammalian metabolism. Emphasis is placed on the potential of microorganisms to mimic mammalian metabolism and provide ways for structural elucidation and toxicological and pharmacological studies of metabolites. Microorganisms can provide difficult-to-synthesize drugs and assist in identifying metabolic pathways of drugs.     

Manufacture of biopharmaceutical proteins by mammalian cell culture systems

In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein. Currently, there are several hundred such candidate drugs in human clinical trials. In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms. The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production. This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually. As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.     

13C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling

¹³C metabolic flux analysis (¹³C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now, ¹³C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for ¹³C-MFA. Specifically, we demonstrate that isotopic labeling of glucose moiety of glycogen and ribose moiety of RNA greatly enhances resolution of metabolic fluxes in the upper part of metabolism; importantly, these measurements allow precise quantification of net and exchange fluxes in the pentose phosphate pathway. To demonstrate the practical importance of these measurements for ¹³C-MFA, we have used Escherichia coli as a model microbial system and CHO cells as a model mammalian system. Additionally, we have applied this approach to determine metabolic fluxes of glucose and xylose co-utilization in the E. coli ΔptsG mutant. The convenience of measuring glycogen and RNA, which are stable and abundant in microbial and mammalian cells, offers the following key advantages: reduced sample size, no quenching required, no extractions required, and GC-MS can be used instead of more costly LC-MS/MS techniques. Overall, the presented approach for ¹³C-MFA will have widespread applicability in metabolic engineering and biomedical research.     

BRCA2-RAD51-DSS1 interplay examined from a microbial perspective

The tumor suppressor BRCA2 plays an essential role in the repair of double-strand DNA breaks by regulating the action of the RAD51 recombinase. The activity of BRCA2 is in turn governed by DSS1, a small acidic protein that appears to function as a necessary cofactor. A model fungal system that reproduces the BRCA2-RAD51 interaction offers the opportunity to understand the mechanism of DSS1 activation at the molecular level.