Alternatives to animal testing in the safety evaluation of products

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.     

Chemical analysis of materials used in pig housing with respect to the safety of products of animal origin

Bedding, environmental enrichment materials and disinfectant powders in pig farming are meant to ensure a hygienic bedding environment or allow pigs to perform explorative behaviour. To our knowledge, no legal regulation exists, that established maximum contents for undesirable substances, such as toxic metals, dioxins or trace elements in these materials, although oral ingestion could be expected. In the present study, a total of 74 materials (disinfectant powders [n = 51], earth/peat [n = 12], biochar [n = 8], recycled manure solids [n = 3]) were analysed for their content of various toxic metals, trace elements, dioxins and polychlorinated biphenyls. The data suggest that, in some samples, trace elements like iron, copper and zinc might have been added intentionally in order to induce physiological effects (iron supply to piglets, copper and zinc as growth promoter in pigs). Moreover, some materials contained high levels of lead, cadmium or arsenic. Consequently, if farm animals repeatedly consume environmental enrichment and bedding materials or disinfectant powders in considerable amounts and these quantities are added to the daily ration, the amount of ingested undesirable substances and trace elements might exceed the maximum levels set for complete feedstuffs, and an elevated transfer into food of animal origin might occur. Future studies are required to address the possible quantitative contribution in the light of feed and food safety. Finally, the excretion of undesirable substances with manure needs to be considered due to their possible accumulation in soils.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

On residues in the disallowed region of the Ramachandran map.

An analysis of the occurrence of nonglycyl residues in conformations disallowed in the Ramachandran plot is presented. Ser, Asn, Thr, and Cys have the highest propensities to exhibit such conformations, and the branched aliphatic residues the lowest. Residues cluster in five regions and there are some trends in the types of residues and their side-chain conformations (chi(1)) occupying these. Majority of the residues are found at the edge of helices and strands and in short loops, and are involved in different types of weak, stabilizing interactions. A structural motif has been identified where a residue in disallowed conformation occurs as the first residue of a short 3(10)-helix. On the basis of the types of neighboring residues, the location in the three-dimensional structure and accessibility, there are similarities with the occurrence of cis peptide bonds in protein structures.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.     

Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

Oxidation of zearalenone by extracellular enzymes from Acinetobacter sp. SM04 into smaller estrogenic products

Zearalenone is a mycotoxin mainly produced by Fusarium mold, which has been associated with hyperestrogenism and other reproductive disorders in pigs, sheep, and other farm animals. Since zearalenone engendered economic losses to farm animal production, its detoxification in contaminated grains or by-products would be advantageous. In this study, enzymes from the Acinetobacter sp. SM04 extracellular extracts of liquid cultures were isolated by Sephadex G-100 column, and an active fraction capable of efficiently degrading zearalenone was obtained. Zearalenone could be oxidized into smaller estrogenic products by the active fraction, and two intermediate products, ZEN-1([M-H]⁻ at m/z 489) and ZEN-2([M-H]⁻ at m/z 405), were found. Zearalenone degradation activity of the active fraction was significantly inhibited by low oxygen gas content, protease, SDS, and EDTA treatment. Further, enzymes in the active fraction were analysed by SDS–PAGE and MALDI-TOF/TOF/MS, and three proteins were found that matched the database for Acinetobacter genus with great homology. They were identified as peroxiredoxin, a possible cytochrome and a putative fimbrial protein precursor.     

A brave new beef: The US food and drug administration's review of the safety of cloned animal products

To meet its public mandate, the US Food and Drug Administration (FDA) collected studies on the potential health hazards of eating or drinking cloned food products. Based on an earlier National Academy of Sciences study that, on closer analysis, was not nearly as sanguine, the FDA's report found no evidence of a health risk from the public's ingestion of cloned food products. This article analyzes the risks the FDA considered, and concludes that there is a disconnect between the risks the FDA assessed in these studies and the risks that might arise from cloned food products. The FDA should consider instituting effective tracking mechanisms and other diagnostics that would permit scientists and the public to answer the question of health risks posed by cloned food products.     

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.     

Gonadotrophin-releasing hormone (GnRH) in the animal kingdom

As a major actor of the brain-pituitary-gonad axis, GnRH has received considerable attention, mainly in vertebrates. Biochemical, molecular, neuroanatomical, pharmacological and physiological studies have mainly focused on the role of GnRH as a gonadotrophin-releasing factor and have led to a detailed knowledge of the hypophysiotrophic GnRH system, primarily in mammals, but also in fish. It is now admitted that the corresponding neurons develop from the olfactory epithelium and migrate into the forebrain during embryogenesis to establish connections with the median eminence in tetrapods or the pituitary in teleost fish. However, all vertebrates possess a second GnRH system, expressing a variant known as chicken GnRH-II in neurons of the synencephalon, whose functions are still under debate. In addition, many fish species express a third form, salmon GnRH, whose expression is restricted to neurons of the olfactory systems and the ventral telencephalon, with extensive projections in the brain and a minor contribution to the pituitary. In vertebrates, GnRHs are also expressed in the gonads where they act on cell proliferation and steroidogenesis in males, and apoptosis of granulosa cells and reinititaion of meiosis in females. These functions could possibly represent the primitive roles of GnRH-like peptides, as an increasing number of studies in invertebrate classes point to a more or less direct connection between GnRH-producing sensory neurons and the gonads. According to recent studies, GnRHs appear as very ancient peptides that emerged at least in the cnidarians, the first animals with a nervous system. GnRH-like peptides have been partially characterized in several classes of invertebrates notably in molluscs, echinoderms and prochordates in which effects on the reproductive functions, notably gamete release and steroidogeneis, have been evidenced. It is possible that, with the increasing complexity of metozoa, GnRH neurons have lost their direct connection with the gonad to specialize in the control of additional regulatory centers such as the hypophysis in vertebrates or the optic gland in cephalopods. However, reminiscent effects of GnRH functions at the gonadal level would have persisted due to local production of GnRHs in the gonad itself. Altogether, these data indicate that GnRHs were involved in the control of reproduction long before the appearance of pituitary gonadotrophs.     

On the existence and separation of the follicle stimulating hormone releasing hormone from the luteinizing hormone releasing hormone.

Porcine hypothalamic fragments were extracted by 2M AcOH at 4°C, and the extractives were subsequently processed in the presence of one protease inhibitor and one anti-oxidant. Gel filtration was performed on Bio-Gel P-2, and supplementary [³H]-LHRH and [¹⁴C]- 3H]-LHRH, and was differentiated from [¹⁴C]- 相似文献