Successful Engraftment of Human Hepatocytes in uPA-SCID and FRG® KO Mice.

Mice with humanized chimeric liver are promising in vivo tools to evaluate the efficacy of novel compounds or vaccine induced antibodies directed against pathogens that infect the human liver. In addition they can be used to study the human-type metabolism of medicinal compounds and hepatotoxicity.

Classification of reproductive toxicants with diverse mechanisms in the embryonic stem cell test.

The embryonic stem cell test (EST) is a promising system to detect embryotoxicity in vitro. Recent studies have pointed out some limitations of the EST and suggest that the applicability domain of the EST and its prediction model have to be better defined. Here, eight substances of known reproductive toxicity were tested in the EST under blind conditions. We applied the prediction model to the data of the EST after classifying the substances according to the published criteria. In addition, a simplified classification of the EST results into two classes as an approach to hazard assessment was compared to the European Union Classification, Labelling and Packaging (CLP) Regulation labels of the substances. With one exception, substances that are labeled as reproductive toxicants according to the CLP Regulation were detected as embryotoxic in the EST while substances without label were found to be non-embryotoxic according to the EST.

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Hyperketonemia (acetoacetate) upregulates NADPH oxidase 4 and elevates oxidative stress, ICAM-1, and monocyte adhesivity in endothelial cells.

BACKGROUND/AIMS: The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D) patients. Hyperketonemia (acetoacetate, ß-hydroxybutyrate) is frequently found along with hyperglycemia in T1D. Whether hyperketonemia per se contributes to the excess oxidative stress and cellular injury observed in T1D is not known. METHODS: HUVEC were treated with ketones in the presence or absence of high glucose for 24 h. NOX4 siRNA was used to specifically knockdown NOX4 expression in HUVEC. RESULTS: Ketones alone or in combination with high glucose treatment cause a significant increase in oxidative stress, ICAM-1, and monocyte adhesivity to HUVEC. Using an antisense approach, we show that ketone induced increases in ROS, ICAM-1 expression, and monocyte adhesion in endothelial cells were prevented in NOX4 knockdown cells. CONCLUSION: This study reports that elevated levels of ketones upregulate NOX, contributing to increased oxidative stress, ICAM-1 levels, and cellular dysfunction. This provides a novel biochemical mechanism that elucidates the role of hyperketonemia in the excess cellular injury in T1D. New drugs targeting inhibition of NOX seems promising in preventing higher risk of complications associated with T1D.

Evidence that 2-methylacetoacetate induces oxidative stress in rat brain.

In the present study we investigated the effects of 2-methylacetoacetate (MAA) and 2-methyl-3-hydroxybutyrate (MHB), the major metabolites accumulating in mitochondrial 2-methylacetoacetyl-CoA thiolase (KT) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies, on important parameters of oxidative stress in cerebral cortex from young rats. We verified that MAA induced lipid peroxidation (increase of thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence values), whereas MHB did not alter these parameters. MAA-induced increase of TBA-RS levels was fully prevented by free radical scavengers, indicating that free radicals were involved in this effect. Furthermore, MAA, but not MHB, significantly induced sulfhydryl oxidation, implying that this organic acid provokes protein oxidative damage. It was also observed that MAA reduced GSH, a naturally-occurring brain antioxidant, whereas MHB did not change this parameter. Furthermore, the decrease of GSH levels caused by MAA was not due to a direct oxidative action, since this organic acid did not alter the sulfhydryl content of a commercial solution of GSH in a cell free medium. Finally, MAA and MHB did not raise nitric oxide production. The data indicate that MAA induces oxidative stress in vitro in cerebral cortex. It is presumed that this pathomechanism may be involved in the brain damage found in patients affected by KT deficiency.

Effects of a novel allelochemical ethyl 2-methyl acetoacetate (EMA) on the ultrastructure and pigment composition of cyanobacterium Microcystis aeruginosa.

Allelochemical ethyl 2-methyl acetoacetate (EMA) can significantly inhibit the growth of bloom-forming Microcystis aeruginosa. In order to assess the implication of the damage of EMA on the algal photosynthetic apparatus, the effects of EMA on the algal ultrastructure and pigment composition were investigated. At initial exposure time to EMA (0-40 h), algal allophycocyanin, phycoerythrin and carotenoid degraded firstly; chlorophyll a increased, especially by 47% in the algae exposed to 2 mg L(-1) of EMA; phycocyanin was not significantly affected; lipid bodies increased remarkably. After 40 h of EMA exposure, chlorophyll a decreased gradually, especially by 45% in the algae exposed to 4 mg L(-1) of EMA; lipid bodies greatly reduced but cyanophycin granules accumulated; thylakoid structures were dissolved or disappeared with the presence of numerous vacuoles. These results showed that all ophycocyanin, phycoerythrin and carotenoid were more sensitive to EMA than other pigments, the cells of M. aeruginosa was stressed by EMA with the occurrence of cyanophycin granules and the photosynthesis pigments and ultrastructure of M. aeruginosa were quickly destroyed by EMA with exposure time increasing.

Time dependence in mixture toxicity with soft electrophiles: 1. Combined effects of selected SN2- and SNAr-reactive agents with a nonpolar narcotic.

Frequently the toxicity of an organic chemical mixture is close to dose-additive, even when the agents are thought to induce toxicity at different molecular sites of action. These findings appear to conflict with the hypothesis that a strictly dose-additive combined effect will be observed for agents sharing a single molecular site of toxic action within the organism. In this study, several SN2-reactive (alpha-halogen) or S(N)Ar-reactive (halogenated dinitrobenzene) soft electrophiles were tested with a model nonpolar narcotic (NPN) to determine the toxicity of the combinations. A sham combination of the model NPN (3-methyl-2-butanone) was also tested as a positive control. The study design incorporated time-dependent toxicity (TDT) determinations at 15, 30, and 45 minutes using a Microtox (Vibrio fischeri) protocol that included testing seven duplicated concentrations for each single agent and mixture per combination. Additionally, in chemico reactivity was determined for each compound using thiol in glutathione as a model nucleophile. The model NPN alone lacked reactivity and TDT. The SN2-reactive agents individually showed varying levels of both reactivity and TDT alone, while the SNAr-reactive chemicals alone were reactive and had toxicity that was fully time-dependent between 15 and 45 minutes of exposure. Data analyses indicated that the sham combination was dose additive, as expected, whereas three of four SN2:NPN combinations showed effects close to that predicted for dose addition but with some differences. The fourth SN2:NPN combination, which included an alpha-halogen with full TDT, showed a less-than-dose-additive combined effect as did both of the SNAr:NPN pairings. By incorporating TDT values, shapes of the dose-response curves, chemical reactivity data with thiol, reactive mechanisms for the soft electrophiles, and quantitative structure activity relationship information on whether the toxicity of the individual soft electrophiles did or did not exceeded that predicted for baseline narcosis, the results suggested that the alpha-halogens elicited two toxic effects at the concentrations tested (reactivity and narcotizing effects), whereas toxicity induced by the halogenated dinitrobenzenes was essentially limited to reactive effects. Collectively, these results provide experimental evidence consistent with previous explanations as to why binary mixtures of industrial organic chemicals often show combined effects that are close to dose additive, even when the chemicals are thought to induce toxicity at different molecular sites of action.

Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors.

Tyrosinemia type 1, caused by mutations in the fumarylacetoacetate hydrolase gene (Fah), is characterized by severe liver injury. We earlier developed a tyrosinemic mouse model with two genetic defects, Fah and 4-hydroxyphenylpyruvate dioxygenase (Hpd) deficiencies. Apoptosis of hepatocytes was induced and an acute onset of liver failure occurred after administration of homogentisic acid (HGA), the intermediate metabolite between the enzymes HPD and FAH. Cytochrome c was released from mitochondria prior to liver failure in the Fah-/- Hpd-/- double-mutant mice after the administration of HGA. In a cell-free system, the addition of fumarylacetoacetate induced the release of cytochrome c from the mitochondria. We also found that caspase inhibitors were highly effective in preventing the liver failure induced by HGA in the double-mutant mice. Therefore, fumarylacetoacetate apparently induces the release of cytochrome c, which in turn triggers activation of the caspase cascade in hepatocytes of subjects with hereditary tyrosinemia type 1.

The mutagenicity of the tyrosine metabolite, fumarylacetoacetate, is enhanced by glutathione depletion.

The toxicity of tyrosine metabolites has been suggested, but not proven, to play a role in the ethiopathogenesis of hepatic alterations observed in hereditary tyrosinemia type I (HT I), a metabolic disease caused by a deficiency of the last enzyme in the tyrosine catabolic pathway, fumarylacetoacetate hydrolase. One of these metabolites, fumarylacetoacetate (FAA), is mutagenic in Chinese hamster V79 cells. We report here that FAA is a powerful glutathione depletor in this cell system. Moreover, the mutagenicity of FAA (100 microM) is potentiated by depletion of cellular glutathione (12% of control levels) by pretreatment with L-buthionine-(S,R)-sulphoximine. In this case, the mutation frequency induced by FAA is 10 times higher than in untreated, control cells. This enhancement is abolished by a partial replenishment of intracellular glutathione (32% of control levels) prior to FAA treatment. Reactive oxygen species are not generated during FAA treatment of glutathione-depleted or undepleted cells. Although the mechanism(s) underlying the mutagenic activity of FAA remains to be identified, these results show that the glutathione depletion activity of FAA may play an important role in the manifestation of its mutagenicity which likely contributes to the HT I-associated liver pathologies.

A 28-day feeding study with ethyl acetoacetate in rats.

Ethyl acetoacetate encapsulated in gum arabic was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 100, 300 and 1000 mg/kg body weight/day. A further group of 16 male and 16 female rats was given rodent diet containing gum arabic as a control. The administration of ethyl acetoacetate in the diet did not adversely affect the growth or general health of the animals or their food intakes. None of the minor variations observed in the haematology, serum chemical analyses or urine analyses are considered to be indicative of a treatment-related toxic effect. Caecal enlargement was seen in male rats treated with the top dose of ethyl acetoacetate, but this was accompanied by a normal histopathology. Few histopathological abnormalities were observed. Proteinaceous casts were found in the bladder of approximately half the male rats given 1000 mg ethyl acetoacetate/kg, and nephrocalcinosis was a common occurrence in female rats in this dose group. Renal function was unimpaired in treated male and female rats, and the histopathological findings are common in the strain of rats chosen for this study. Although the caecal enlargement and the changes in kidney and bladder of rats given 1000 mg ethyl acetoacetate/kg are noted, it is considered that ethyl acetoacetate did not produce treatment-related adverse effects in rats during this study.

[The establishment of the MPEL for carboxylic acid derivatives in the air of a work area by using mathematical models]. / Obosnovanie PDK nekotorykh proizvodnykh karbonovykh kislot v vozdukhe rabochei zony s ispol'zovaniem matematicheskikh modelei.

Reports of MACs for carboxylic acid derivatives as sodium methoxyacetate (SMOA), methyl chlorpropionate (MCP) and acetoacetic ether (AAE). SMOA shows low toxicity, no cumulation and specific action MCP demonstrates moderate toxicity, low cumulation no specific action in concentrations close to Limac. AAE presents toxicity low in oral usage and moderate when inhaled, low cumulation, no irritation and sensitization. Tentative safe level of injury and MAC estimated for the aforesaid substances together with toxicity parameters compared with the substances studied before allowed the authors to set the MAC of SMOA--10, MCP--2, AAE--10 mg/m3.

Effects of hyperglycemia and ketone bodies on the in vitro development of early somite rate embryos.

The teratogenic effects of diabetes are attributed to the influence of hyperglycemia and hyperketonemia as well as to other metabolic factors. We studied the effects of ketone bodies and glucose on the development of early somite rat embryos (day 10 1/2 of gestation) cultured in vitro. D-glucose was added to normal rat serum in concentrations of 2, 5, and 10 mg/ml. Ketone bodies (acetoacetate and beta-hydroxybutyrate, B-HOB) were individually added to normal or to hyperglycemic sera (total glucose concentrations of 3 mg/ml) in the following concentrations: acetoacetate--5, 10, 20, and 40 micrograms/ml; B-HOB--2, 5, and 8 mg/ml. The higher concentrations of each of the substances induced growth retardation and abnormalities. The growth-retarding and teratogenic effects of a combination of the substances, glucose and B-HOB, glucose and acetoacetate, on the development of 10 1/2 day embryos were greater than when each substance was added separately, even at relatively low doses. The greatest teratogenic effects were observed when low concentrations of all three substances were added simultaneously to the culture medium. These results may have direct relevance to human diabetes since diabetes is characterized by a simultaneous elevation of serum levels of all these substances.