Animal Bioassays Research Articles

Biological methods for marine toxin detection

The presence of marine toxins in seafood poses a health risk to human consumers which has prompted the regulation of the maximum content of marine toxins in seafood in the legislations of many countries. Most marine toxin groups are detected by animal bioassays worldwide. Although this method has well known ethical and technical drawbacks, it is the official detection method for all regulated phycotoxins except domoic acid. Much effort by the scientific and regulatory communities has been focused on the development of alternative techniques that enable the substitution or reduction of bioassays; some of these have recently been included in the official detection method list. During the last two decades several biological methods including use of biosensors have been adapted for detection of marine toxins. The main advances in marine toxin detection using this kind of technique are reviewed. Biological methods offer interesting possibilities for reduction of the number of biosassays and a very promising future of new developments.

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers

Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR CT values between Wescodyne®, acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox® resulted in a 2-log10 reduction in CT values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne® treated oocysts while at least a 2-log10 reduction in plaques formed was observed with Clorox® treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log10 reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

Potential for development of an Escherichia coli-based biosensor for assessing bioavailable methionine: a review.

Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs.

BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures

The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20years, however, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, animal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcinogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008).The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a modified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treatment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suitability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans.

Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides

Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides

Commentary on ‘‘Toxicity Testing in the 21st Century: A vision and a Strategy’’

The vision of the NAS Committee represents a transition in toxicology from an emphasis on the study of apical endpoints in whole animals to a focus on the study of pathway perturbations, based primarily on in vitro data, from which dose-response models will ultimately be developed. It should be emphasized that this vision does not seek to eliminate whole-animal testing altogether; however, it does aim to eventually use animal testing to a far less extent than in our current system. Such a change to the current method of toxicity testing represents an important goal for many reasons. For example, toxicity testing needs to incorporate the knowledge that has been developed through the most recent advances in research, particularly at the molecular level. Through the application of mechanistic information resulting from the testing of chemicals by the proposed in vitro methods, it may be possible to predict the toxicity of similar chemicals that have not yet been tested in whole animals. The number of chemicals in commercial use for which we have adequate toxicity information is limited, and higherthroughput assays have the potential to increase this number. While the vision’s approach would result in a loss of information from a reduction in testing at high doses, the NAS Committee has proposed that this lack of information would be compensated by a greater understanding of effects at low doses, and that such an understanding can eventually be applied in a practical context. Clearly, however, there are multiple challenges to the actual implementation of the vision. First, we need to consider the vast biological complexity of whole organisms. Will in vitro assays for pathway perturbations, in the absence of some understanding from in vivo studies, be able to identify all of the possible toxic endpoints of a particular chemical? The application of in vitro methods may be a slow and uncertain process without some initial hypotheses about likely critical apical endpoints for a particular chemical. In contrast, with animal bioassays, we can identify toxic endpoints that we may not have originally thought to look for, through the analysis of different target organs. An understanding of how pathway activation and the corresponding dose-response relationships change with differences in the duration of exposure will be necessary, and yet it may be difficult to distinguish between acute, subchronic, and chronic effects at the pathway level. In addition, in vitro assays cannot mirror the metabolism of a whole animal, and the NAS Committee has agreed that much research will be needed to ensure that the new testing methods associated with their vision fully evaluate the effects of both chemicals and their metabolites. The development of ‘-omic’ technologies (such as genomics, proteomics, and metabolomics) is a key element for advancing the vision of the NAS Committee; however, there are challenges to the use of these approaches for the prediction of in vivo toxicity. Rhomberg et al. state that:

Genotoxic evaluation of an industrial effluent from an oil refinery using plant and animal bioassays

Polycyclic aromatic hydrocarbons (PAHs) are genotoxic chemicals commonly found in effluents from oil refineries. Bioassays using plants and cells cultures can be employed for assessing environmental safety and potential genotoxicity. In this study, the genotoxic potential of an oil refinery effluent was analyzed by means of micronucleus (MN) testing of Alium cepa, which revealed no effect after 24 h of treatment. On the other hand, primary lesions in the DNA of rat (Rattus norvegicus) hepatoma cells (HTC) were observed through comet assaying after only 2 h of exposure. On considering the capacity to detect DNA damage of a different nature and of these cells to metabolize xenobiotics, we suggest the association of the two bioassays with these cell types, plant (Allium cepa) and mammal (HTC) cells, for more accurately assessing genotoxicity in environmental samples.

Factors to consider in the use of stem cells for pharmaceutic drug development and for chemical safety assessment

Given the reality of the inadequacies of current concepts of the mechanisms of chemical toxicities, of the various assays to predict toxicities from current molecular, biochemical, in vitro and animal bioassays, and of the failure to generate efficacious and safe chemicals for medicines, food supplements, industrial, consumer and agricultural chemicals, the recent NAS Report, “ Toxicity Testing in the 21st Century: A Vision and a Strategy”, has drawn attention to a renewed examination of what needs to be done to improve our current approach for better assessment of potential risk to human health. This “ Commentary” provides a major paradigm challenge to the current concepts of how chemicals induce toxicities and how these various mechanisms of toxicities can contribute to the pathogenesis of some human diseases, such as birth defects and cancer. In concordance with the NAS Report to take “… advantage of the on-going revolution in biology and biotechnology”, this “Commentary” supports the use of human embryonic and adult stem cells, grown in vitro under simulated “in vivo niche conditions”. The human being should be viewed “as greater than the sum of its parts”. Homeostatic control of the “emergent properties” of the human hierarchy, needed to maintain human health, requires complex integration of endogenous and exogenous signaling molecules that control cell proliferation, differentiation, apoptosis and senescence of stem, progenitor and differentiated cells. Currently, in vitro toxicity assays (mutagenesis, cytotoxicity, epigenetic modulation), done on 2-dimensional primary rodent or human cells (which are always mixtures of cells), on immortalized or tumorigenic rodent or human cell lines do not represent normal human cells in vivo [which do not grow on plastic and which are in micro-environments representing 3 dimensions and constantly interacting factors]. In addition, with the known genetic, gender, and developmental state of cells in vivo, any in vitro toxicity assay will need to mimic these conditions in vitro. More specifically, while tissues contain a few stem cells, many progenitor/transit cells and terminally differentiated cells, it should be obvious that both embryonic and adult stem cells would be critical “target” cells for toxicity testing. The ultimate potential for in vitro testing of human stem cells will to try to mimic a 3-D in vitro micro-environment on multiple “organ-specific and multiple genotypic/gender “adult stem cells. The role of stem cells in many chronic diseases, such as cancer, birth defects, and possibly adult diseases after pre-natal and early post-natal exposures (Barker hypothesis), demands toxicity studies of stem cells. While alteration of gene expression (“toxico-epigenomics”) is a legitimate endpoint of these toxicity studies, alteration of the quantity of stem cells during development must be serious considered. If the future utility of human stem cells proves to be valid, the elimination of less relevant, expensive and time-consuming rodent and 2-D human in vitro assays will be eliminated.

Continuous Quinacrine Treatment Results in the Formation of Drug-Resistant Prions

Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy in vivo. As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR0/0 mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 µM of quinacrine in their brains without acute toxicity. PrPSc levels in the brains of prion-inoculated MDR0/0 mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrPSc levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR0/0 mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrPSc levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrPSc that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR0/0 mice. From these data, we propose that quinacrine eliminates a specific subset of PrPSc conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs.

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than ∼15 nm in diameter).

Applications of the Poly-K Statistical Test to Life-Time Cancer Bioassay Studies

ABSTRACT The statistical analysis of cancer bioassay data has historically depended on the pathological determination of the experimental animal's cause of death. The poly-k statistical test has provided a method of statistical analysis of animal bioassay data without the need for cause of death information. The test has been shown to have good statistical properties in the typical 2-year cancer bioassay. However, while the poly-k test has been applied to chronic lifetime animal studies, it has not been formally evaluated with respect to the operating characteristics of this statistical test when applied to such studies. Thus, our objective is to assess the performance of the poly-k test for lifetime studies and to make comparisons with other tests. We observed in one recent lifetime study of the gasoline additive methyl tertiary butyl ether (MTBE) that the application of the poly-k test was not statistically robust. Simulation studies were subsequently conducted for a limited number of scenarios of lifetime cancer bioassays. These simulations showed that the poly-k test is not statistically robust for testing effect of increasing dose in some lifetime cancer studies.

Modulation of Anticancer Drug-Induced P-Glycoprotein Expression by Naringin

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in the tumour cells of a patient, resulting from enhanced drug efflux. It is often related to the overexpression of P-glycoprotein (P-gp) on the surface of tumour cells, thereby reducing drug cytotoxicity. In the present study, naringin (the predominant flavonone found in grapefruit and other related citrus species) was tested for its potential ability to modulate the expression of P-gp in a short-term animal bioassay, in comparison with verapamil (a calcium channel blocker and positive MDR reversal agent). Western blot analysis showed that pre-treatment by i.p. administration of 5 mg naringin/kg body weight for 3 consecutive days prior to doxorubicin (the most common used anticancer drug which induces MDR) administration was able to significantly lower the P-gp expression reaching nearly the level of animals treated with verapamil. Moreover, pre-treatment with naringin prior to doxorubicin increased the sensitivity to the drug. Naringin inhibited the doxorubicin-stimulated ATPase activity demonstrating that naringin may interact directly with the transporter. In addition, the results demonstrated that induction of both glutathione (GSH) and glutathione-S-transferase (GST) by doxorubicin is consistent with an increased ATP-dependent doxorubicin transport. Thus, naringin seems to modulate the in vivo expression of P-gp. In summary, the present study describes the dual modulation of P-gp expression and function by the flavonoid naringin, which may be an attractive new agent for the chemosensitization of cancer cells.

Lead toxicity, vulnerable subpopulations and emergency preparedness

This paper reviews some evidence of the toxic effects of lead (Pb) in the context of vulnerable subpopulations and emergency preparedness. Pb is ubiquitous in the environment and is used in many building materials. Environmental Pb concentrations and body burdens of Pb have been shown to increase following disasters. Pb is a systemic toxicant with no known beneficial biological function and, for several endpoints, no identified threshold of toxicity. The fetus, children, pregnant and elderly are particularly susceptible to some of the toxic effects of Pb. Pb exposures in infancy have been shown in vivo to produce an equal degree of neurobehavioural impairment as exposures of much longer duration at equivalent doses during adolescence. Evidence from animal bioassays indicates that the carcinogenic potency of perinatal Pb exposure may be about 3-fold higher than adult lifetime exposure at an equivalent dose. Animal assays show up to a 12-fold difference between fetal, neonatal and adult life stages in sensitivity to the immunological effects of Pb. Pb exposure is associated with increased risk of cardiovascular and cerebrovascular morbidity and mortality--health endpoints for which the elderly are at increased risk. Finally, endogenous and exogenous variables, such as psychological and physiological stress, dietary deficiencies and concomitant exposure to other chemical, biological and radiological hazards, can also potentially modify or potentiate the toxic effects of Pb. Because of the potential for concurrent influence of these modifying variables in a post-disaster environment, emergency response planners are encouraged to consider disaster victims and responders, as a whole, as a potentially vulnerable subpopulation.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100x higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Monitoring toxicity of an azo dye methyl red and a heavy metal Cu, using plant and animal bioassays

Toxicities of an azo dye methyl red and a heavy metal copper (Cu) were quantified, using growth and mortality as end points, in four plant species and three animal species by subjecting them to short-term (4 days for animals, 10 days for plants) static bioassays. Lemna aequinoctialis Welwitch (EC50: 7–16 ppm) was found to be the most sensitive species for methyl red, Ceratophyllum demersum L. (EC50: 25 ppm) and Lactuca sativa L. (EC50: 56 ppm) were intermediate, while Oryza sativa L. shows reduction in seedling vigor (9–27%) of <50%, being the least sensitive amongst the tested plant species. Methyl red toxicity is almost 3–5-fold higher in growing medium (pH = 5.8–6.0), even at high nutrient levels, while Cu toxicity is higher in nutrient-poor alkaline medium at alkaline pH (8.3–8.7; EC50: Ceratophyllum = 104–200 ppb; Lemna = 100–170 ppb) compared to nutrient-rich acidic medium (pH = 5.4–5.7; EC50: Ceratophyllum = 2600–3175 ppb; Lemna = 4350–4715 ppb). Rice tolerance (EC50: 6500 ppb) was found to be higher than hydrophytes while lettuce was most tolerant to Cu. Fish sensitivity toward the test chemicals was almost parallel to Ceratophyllum and Lemna [Gambusia affinis Baird and Gerard (LC50: 250 ppb for Cu) and Poecilia reticulata Peters (LC50: 24 ppm for methyl red)]. Similar to the plants, dye toxicity increased markedly (LC50: 7 ppm) in the acidic medium (pH = 6.0). Amongst the tested organisms, Daphnia was found to be most sensitive to methyl red (EC50: 6 ppm) while its sensitivity to Cu (EC50: 230 ppb) was similar to hydrophytes and fish. Initially, the combination of dye and Cu (at their sublethal concentrations) had additive effects in duckweed, while dye concentration ruled afterward. These results indicate that hydrophytes and animals are equally sensitive toward the test chemicals. Dye toxicity in hydrophytes and fish was pH dependent, while in the case of Cu, it is related to the nutrient status of the growth medium of plants.

Advances in chemical carcinogenesis: a historical review and prospective.

The American Association for Cancer Research has been the citadel for communicating research on chemical carcinogens for over a century. It therefore seems appropriate that a review of chemical carcinogenesis inaugurates a series of articles highlighting advances in understanding, treating, and

A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development

BackgroundThe alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish.MethodsZebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf) and continuing with daily refreshing until 7 dpf.ResultsPPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in C. elegans support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics.ConclusionOwing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models.

Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process.

Uncertainties in biologically-based modeling of formaldehyde-induced respiratory cancer risk: identification of key issues.

In a series of articles and a health-risk assessment report, scientists at the CIIT Hamner Institutes developed a model (CIIT model) for estimating respiratory cancer risk due to inhaled formaldehyde within a conceptual framework incorporating extensive mechanistic information and advanced computational methods at the toxicokinetic and toxicodynamic levels. Several regulatory bodies have utilized predictions from this model; on the other hand, upon detailed evaluation the California EPA has decided against doing so. In this article, we study the CIIT model to identify key biological and statistical uncertainties that need careful evaluation if such two-stage clonal expansion models are to be used for extrapolation of cancer risk from animal bioassays to human exposure. Broadly, these issues pertain to the use and interpretation of experimental labeling index and tumor data, the evaluation and biological interpretation of estimated parameters, and uncertainties in model specification, in particular that of initiated cells. We also identify key uncertainties in the scale-up of the CIIT model to humans, focusing on assumptions underlying model parameters for cell replication rates and formaldehyde-induced mutation. We discuss uncertainties in identifying parameter values in the model used to estimate and extrapolate DNA protein cross-link levels. The authors of the CIIT modeling endeavor characterized their human risk estimates as "conservative in the face of modeling uncertainties." The uncertainties discussed in this article indicate that such a claim is premature.

NUTRITIONAL AND FUNCTIONAL PROPERTIES OF JOJOBA, HIBISCUS AND PUMPKIN SEED KERNAL FLOURS

The, present study was carried out to maximizing the available and valuable sources of proteins of the agriculture plant`s waste and industry duties . So this research focusing on the nutritional and functional properties of defatted hibiscus, jojoba and pumpkin seeds flour. Analysis were carried out on defatted seeds, to determine proximate chemical composition , such as crude protein , ether extract, ash, crude fiber, amino acids and carbohydrate content.The latter was calculated by difference. Protein content ranged of 23.4, 28.0 and 53.5 % of dry mater base (DM) of jojoba seeds flour (JF), hibiscus seeds flour (HF), and pumpkin seeds flours (PF) respectively. In addition, fat content ranged from 13.9 % to 33.8 % of HF and PF, respectively, while the highest fat content 54.9% obtained from JF. Antinutritional compounds, such as phytates detected in all flours, ranged from 0.23 to 0.5 g/100g DM. Functional properties of all flours were assessed, such as water absorption index (WAI), water soluble index(WSI), acid number (pH), foam capacity (FC) and foam stability (FS). All flours were excellent in their functional properties except FC and FS of PF was inferior good. Amino acids compositions of these flours were also determined. The protein of these flour had abundant amount of total essential amino acids and higher than recommended by FAO/ WHO (1973). This research classified the amino acids of all seeds flour into groups to identify the flour potential for food supplementation in bakery and meat processed formulation. The nutritional value of the protein and its quality methods based on in vitro (chemical) and animal bioassays in vivo for assessment of protein quality have been developed by predicting protein quality depending on the determination of amino acids and calculated chemical scores, essential amino acid index (EAAI), amino acid / essential amino acids ( A/E ) ratio, amino acids score (AAS), estimated biological (BV) and calculated protein efficiency ratio ( PER ). An in vivo of protein quality depending on biological assessment using rats which depending on true digestibility percentage (TD), BV and net protein utilization (NPU) were investigated. By comparison, between their chemical estimation and bioassay, it's clearly view that, both of HF and PF had good quality protein; as expressed by their high TD, BV and NPU when compared to poorly JF. Both of these flours were significantly a quite similar to standard protein of casein. Meanwhile, jojoba flour had a considerable amount of antinutritional factors of simmondsin, which act as satiety inducers and impede bioavailability of protein. All of these nutritional values of the different flours HF and PF concluded those have an excellent protein and nourished sources. These flours could be potentially added to food system, such as bakery products, complementary foods and meat products, moreover JF have been promising not only their nutritive value but also for their responsibility to diminish satiety and reducing food intake for dietetic persons.