Gestational and lactational exposure to the polychlorinated biphenyl mixture Aroclor 1254 modulates retinoid homeostasis in rat offspring.

Polychlorinated biphenyls (PCBs) induce a broad spectrum of biochemical and toxic effects in mammals including alterations of the vital retinoid (vitamin A) system. The aim of this study was to characterize alterations of tissue retinoid levels in rat offspring and their dams following gestational and lactational exposure to the PCB mixture Aroclor 1254 (A1254) and to assess the interrelationship of these changes with other established sensitive biochemical and toxicological endpoints. Sprague-Dawley rat dams were exposed orally to 0 or 15 mg/kg body weight/day of A1254 from gestational day 1 to postnatal day (PND) 23. Livers, kidneys and serum were collected from the offspring on PNDs 35, 77 and 350. Tissue and serum retinoid levels, hepatic cytochrome P450 (CYP) enzymes and serum thyroid hormones were analyzed. A multivariate regression between A1254 treatment, hepatic retinoid levels, hepatic CYP enzymes activities, thyroid hormone levels and body/liver weights was performed using an orthogonal partial least-squares (PLS) analysis. The contribution of dioxin-like (DL) components of A1254 to the observed effects was also estimated using the toxic equivalency (TEQ) concept. In both male and female offspring short-term alterations in tissue retinoid levels occurred at PND35, i.e. decreased levels of hepatic retinol and retinoic acid (RA) metabolite 9-cis-4-oxo-13,14-dihydro-RA with concurrent increases in hepatic and renal all-trans-RA levels. Long-term changes consisted of decreased hepatic retinyl palmitate and increased renal retinol levels that were apparent until PND350. Retinoid system alterations were associated with altered CYP enzyme activities and serum thyroid hormone levels as well as body and liver weights in both offspring and dams. The estimated DL activity was within an order of magnitude of the theoretical TEQ for different endpoints, indicating significant involvement of DL congeners in the observed effects. This study shows that tissue retinoid levels are affected both short- and long-term by developmental A1254 exposure and are associated with alterations of other established endpoints of toxicological concern.

In utero and lactational exposure to a mixture of environmental contaminants detected in Canadian Arctic human populations alters retinoid levels in rat offspring with low margins of exposure.

Arctic inhabitants are highly exposed to persistent organic pollutants (POP), which may produce adverse health effects. This study characterized alterations in tissue retinoid (vitamin A) levels in rat offspring and their dams following in utero and lactational exposure to the Northern Contaminant Mixture (NCM), a mixture of 27 contaminants including polychlorinated biphenyls (PCB), organochlorine (OC) pesticides, and methylmercury (MeHg), present in maternal blood of the Canadian Arctic Inuit population. Further, effect levels for retinoid system alterations and other endpoints were compared to the Arctic Inuit population exposure and their interrelationships were assessed. Sprague-Dawley rat dams were dosed with NCM from gestational day 1 to postnatal day (PND) 23. Livers, kidneys and serum were obtained from offspring on PND35, PND77, and PND350 and their dams on PND30 for analysis of tissue retinoid levels, hepatic cytochrome P-450 (CYP) enzymes, and serum thyroid hormones. Benchmark doses were established for all endpoints, and a partial least-squares regression analysis was performed for NCM treatment, hepatic retinoid levels, CYP enzyme induction, and thyroid hormone levels, as well as body and liver weights. Hepatic retinoid levels were sensitive endpoints, with the most pronounced effects at PND35 though still apparent at PND350. The effects on tissue retinoid levels and changes in CYP enzyme activities, body and liver weights, and thyroid hormone levels were associated and likely driven by dioxin-like compounds in the mixture. Low margins of exposure were observed for all retinoid endpoints at PND35. These findings are important for health risk assessment of Canadian Arctic populations and further support the use of retinoid system analyses in testing of endocrine-system-modulating compounds.

Valproic acid and its derivatives enhanced estrogenic activity but not androgenic activity in a structure dependent manner.

Steroid hormones affect metabolic pathways and cellular functions. Valproic acid (VPA), used as antiepileptic drug, inhibits histone deacetylases and interacts with intracellular receptors. We analyzed the impact of VPA and VPA derivatives on activation of estrogen and androgen receptors (ER and AR) using reporter gene assays. VPA and its long-chain derivatives 2-(2-propynyl)-hexanoic acid [butyl-4-yn-VPA], 2-(2-propynyl)-heptanoic acid [S-pentyl-4-yn-VPA] and 2-(2-propynyl)-nonanoic acid [heptyl-4-yn-VPA] enhanced 17ß-estradiol-induced ERα and ERß activation partly synergistically with a structure-activity correlation. The extent of this effect regarding to ERα activation increased with prolongation of the aliphatic side chain. Regarding AR activation, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA slightly induced AR activity when tested alone. In combination with the AR agonist 5α-dihydrotestosterone, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA showed anti-androgenic effects without an apparent structural relation. Our results indicate that VPA and its derivatives affect estrogen signaling with a structural specificity, while the (anti-)androgenic effects of these compounds are not structurally correlated.

Enantioselective apoptosis induction in histiocytic lymphoma cells and acute promyelocytic leukemia cells.

The aim of this study was to identify valproic acid (VPA) analogs with a broad spectrum of anti-cancer activities and an increased apoptosis-inducing potential compared with the parent VPA, which is enrolled as histone deacetylase (HDAC) inhibitor in a large number of clinical trials. We identified a chiral VPA derivative, (S)-2-pentyl-4-pentynoic acid, previously characterized as HDAC inhibitor that induced massive programmed cell death in a strongly enantioselective manner in U937 histiocytic lymphoma cells and NB4 acute promyelocytic leukemia cells. By performing fluorescence-activated cell sorting and Western blotting analyses, we established that enantiomer (S)-2-pentyl-4-pentynoic acid has higher apoptosis-inducing potential than VPA itself. The optic antipode (R)-2-pentyl-4-pentynoic acid and VPA caused under the same conditions only a weak growth inhibition without inducing cell differentiation and apoptosis. (S)-2-pentyl-4-pentynoic acid is more apoptogenic than VPA and displays enantioselective anti-cancer properties that warrant further research regarding the mechanistic basis of its activity and its potential use in cancer therapy.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of ß-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

The teratogenic potencies of valproic acid derivatives and their effects on biological end-points are related to changes in histone deacetylase and Erk1/2 activities.

Valproic acid (VPA) is a known teratogen. In the present study, the effects of VPA and seven VPA derivatives with different teratogenic potencies (isobutyl-, 5-methyl-, ethyl-, propyl-, butyl-, pentyl- and hexyl-4-yn-VPA) were investigated in L929 cells in vitro. Evaluated end-points included changes in cell proliferation, growth, cell cycle distribution, morphology, speed, glycogen synthase kinase-3ß (GSK-3ß) and Erk1/2 phosphorylation, and histone H3 acetylation. Changes in proliferation, growth, speed, Erk1/2 and GSK-3ß-Tyr216 phosphorylation, and H3 acetylation were significantly associated with the teratogenic potencies of the VPA derivatives. However, in contrast to changes in Erk1/2 phosphorylation and H3 acetylation, significant changes in GSK-3ß phosphorylation could only be obtained in response to prolonged incubation at high drug concentration. There was an association between changes in H3 acetylation and GSK-3ß-Tyr216 phosphorylation, whereas none of these end-points were associated with changes in Erk1/2 phosphorylation. These results suggest that the teratogenic potencies of VPA and VPA derivatives are related to effects on both Erk1/2 and histone deacetylase activities, whereas changes in GSK-3ß activity are possibly a secondary effect.

The embryonic stem cell test as tool to assess structure-dependent teratogenicity: the case of valproic acid.

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.

CYP26A1-specific antagonist influence on embryonic implantation, gene expression and endogenous retinoid concentration in rats.

Retinoids are essential in vertebrate reproduction and embryonic development. All-trans-retinoic acid (ATRA) is tightly regulated during these processes. CYP26A1 is mainly responsible for its degradation. To study the role of CYP26A1 during implantation, we applied R115866, a CYP26A1-specific antagonist, to rats during early gestation days (GD). On GD 6.5 and 12 samples were collected and the number of embryos was evaluated. ATRA concentration increased in uterus and serum, mRNA expression of CYP26A1 and CRABP2 increased in the liver, but not in the uterus. Uterine COX1 and 17ßHSD mRNA expression was decreased. The number of embryos on GD 12 was not altered in this setting. It can be concluded that uterine expression of the analyzed retinoid-response genes during early gestation is not altered by this R115866 treatment and instead indirectly via ATRA. From our experiment we cannot confirm that ATRA obtains a major influencing role in the regulation of embryonic implantation.

Potent neuroprotective effects of novel structural derivatives of valproic acid: potential roles of HDAC inhibition and HSP70 induction.

Emerging evidence suggests that the neuroprotective effects of valproic acid (VPA) occur via inhibition of histone deacetylases (HDACs) and activation of gene expression. This study assessed the ability of four VPA derivatives to cause histone hyperacetylation and protect against glutamate-induced excitotoxicity in cultured neurons. We found that (S)-2-pentyl-4-pentynoic acid (compound III) and (+/-)-2-hexyl-4-pentynoic acid (compound V) were far more potent and robust than VPA in inducing histone hyperacetylation and protecting against glutamate excitotoxicity. Thus, the increase in histone acetylation elicited by compounds III and V was significant at 5microM and reached a maximal increase of 600-700% at 50-100microM, compared with only a 200% increase by VPA at 100microM. The neuroprotective effects of compounds III and V were evident at 10-25microM and reached a complete protection at 50-100microM, while a significant partial protection by VPA was observed at 100microM. These two compounds were also more effective than VPA in increasing HSP70-1a and HSP70-1b mRNA levels. At 50microM, compound V was most robust in increasing HSP-1a mRNA levels, followed by compound III, and then by VPA. HSP-1b mRNA was only significantly upregulated by compounds V and III, but not by VPA or other VPA derivatives under these treatment conditions. Our results suggest that these two VPA derivatives may ultimately be developed into potent neuroprotective drugs in preclinical and clinical studies.

Determination of drug residues in urine of dogs receiving anti-cancer chemotherapy by liquid chromatography-electrospray ionization- tandem mass spectrometry: is there an environmental or occupational risk?

Cytotoxic drugs, previously used only in human medicine, are increasingly utilized for cancer treatment in veterinary practice. We developed and validated a liquid chromatography (LC)-electrospray ionization-tandem mass spectrometry (MS-MS) method to determine vincristine, vinblastine, cyclophosphamide, and doxorubicin in canine urine. Sample pretreatment consisted of liquid-liquid extraction, and LC separation was carried out on an RP C(18) column employing a 0.5% formic acid/methanol gradient system. The analytes were detected in positive ion mode using the MS-MS scan mode. The mean recoveries in six different urine samples were between 64.2% and 86.9%. Limits of quantitation were 0.5 microg/L for vincristine and vinblastine, 1 microg/L for cyclophosphamide, and 5 microg/L for doxorubicin; limits of detection were approximately 0.25 microg/L for vincristine, vinblastine, and cyclophosphamide and 0.5 microg/L for doxorubicin. It could be demonstrated that all investigated drugs are found in urine of dogs undergoing chemotherapy. In samples from day 1 after chemotherapy, as much as 63 microg/L vincristine, 111 microg/L vinblastine, and 762 microg/L doxorubicin could be detected. Cyclophosphamide showed only minor concentrations on day 1, but up to 2583 microg/L could be found directly after chemotherapy. These initial data show that there might be a potential contamination risk when administering cytotoxics in veterinary medicine.

Metabolic activation capacity by primary hepatocytes expands the applicability of the embryonic stem cell test as alternative to experimental animal testing.

The murine embryonic stem cell test (EST) represents a validated alternative method for in vivo embryotoxicity testing. In the present study, primary hepatocytes were combined with the EST by a preincubation approach to improve its predictivity on bioactivation caused teratogenicity. As substances the well-known proteratogens cyclophosphamide (CPA) and valpromide (VPD) were used. The embryotoxic potential of CPA was detected by a strong decrease of the resulting ID(50)-concentration (50% inhibition of ES cell differentiation) after incubation with murine hepatocytes. Interspecies variation in metabolism was detected by testing VPD. After incubation of VPD with murine hepatocytes no inhibition of ES cell differentiation was observed, since hardly any teratogenic VPD metabolites were formed. In contrast, with human hepatocytes a significant conversion of VPD into the teratogen valproic acid (VPA) was observed. In summary we developed a co-culture approach for embryotoxicity testing, whereby the test compounds were incubated with hepatocytes and the supernatant was added to the ES cell culture to obtain a dose dependency of the preincubated test substances.

The endogenous retinoid metabolite S-4-oxo-9-cis-13,14-dihydro-retinoic acid activates retinoic acid receptor signalling both in vitro and in vivo.

Retinoic acid receptor (RAR) and retinoid X receptor are ligand-induced transcription factors that belong to the nuclear receptor family. The receptors are activated by small hydrophobic compounds, such as all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Interestingly, these receptors are also targets for a number of exogenous compounds. In this study, we characterized the biological activity of the 9-cis-substituted retinoic acid metabolite, S-4-oxo-9-cis-13,14-dihydro-retinoic acid (S-4o9cDH-RA). The endogenous levels of this metabolite in wild-type mice and rats were found to be higher than those of all-trans-retinoic acid, especially in the liver. Using cell-based luciferase reporter systems, we showed that S-4o9cDH-RA activates the transcription of retinoic acid response element-containing genes in several cell types, both from a simple 2xDR5 element and from the promoter of the natural retinoid target gene RARbeta2. In addition, quantitative RT-PCR analysis demonstrated that S-4o9cDH-RA treatment significantly increases the endogenous mRNA levels of the RAR target gene RARbeta2. Utilizing a limited proteolytic digestion assay, we showed that S-4o9cDH-RA induces conformational changes to both RARalpha and RARbeta in the same manner as does all-trans-retinoic acid, suggesting that S-4o9cDH-RA is indeed an endogenous ligand for these receptors. These in vitro results were corroborated in an in vivo system, where S-4o9cDH-RA induced morphological changes similar to those of all-trans-retinoic acid in the developing chicken wing bud. When locally applied to the wing bud, S-4o9cDH-RA induced digit pattern duplications in a dose-dependent fashion. The results illustrate that S-4o9cDH-RA closely mimics all-trans-retinoic acid with regard to pattern respecification. Finally, using quantitative RT-PCR analysis, we showed that S-4o9cDH-RA induces the transcription of several retinoic acid-regulated genes in chick wing buds, including Hoxb8, RARbeta2, shh, Cyp26 and bmp2. Although S-4o9cDH-RA was less potent when compared with all-trans-retinoic acid, the findings clearly demonstrate that S-4o9cDH-RA has the capacity to bind and activate nuclear retinoid receptors and regulate gene transcription both in vitro and in vivo.

Hydroxamic acid and fluorinated derivatives of valproic acid: anticonvulsant activity, neurotoxicity and teratogenicity.

PURPOSE: Fluorinated and non-fluorinated valproic acid (VPA) analogues with hydroxamic acid moieties were tested for their teratogenic, anticonvulsant and neurotoxic potencies in mice. METHODS: Compounds were synthesized from their corresponding acids. The induction of neural tube defects (exencephaly) of the resulting hydroxamates (applied on day 8.25 of gestation) was tested in the offspring of pregnant animals (Han:NMRI mice). The anticonvulsant activity was evaluated in the subcutaneous pentylenetetrazole (PTZ) seizure threshold test and neurotoxicity in the rotorod neurotoxicity test. RESULTS: All tested hydroxamates showed no or greatly reduced teratogenic potency in mice compared to the free acids. Furthermore all compounds exhibited anticonvulsant activity with ED(50) doses ranging from 0.16 mmol/kg to 0.59 mmol/kg (VPA 0.57 mmol/kg). Neurotoxicity of the hydroxamates was increased compared to VPA. TD(50) doses range from 0.70 mmol/kg to 1.42 mmol/kg (VPA 1.83 mmol/kg). CONCLUSION: Hydroxamic acid derivatives of VPA with improved protective index and little or undetectable teratogenic potency compared to the free acids are described. alpha-fluorination of VPA also resulted in loss of teratogenic activity. Such fluorination of the hydroxamic acids also led to compounds with an improved anticonvulsant profile compared to non-fluorinated hydroxamates. The non-chiral 2-Fluoro-VPA-hydroxamic acid was the most promising compound with a protective index (ratio of TD(50) to ED(50)) of 4.4 compared to 3.2 for VPA. This compound combines an improved ratio of anticonvulsant potency/neurotoxicity with the advantage of not being teratogenic in the mouse neural tube defect model used.

Penetration studies of propoxur and phoxim from eggshell into whole egg after experimental exposure and application in henhouses.

The penetration of propoxur and phoxim from eggshell into whole egg was investigated in vitro by spraying eggs directly and in vivo after application of the compounds in henhouses. Although mean concentrations of the compounds on eggshells were up to 23000 microg kg(-1), mean residue concentrations in whole eggs were far below the current maximum residue levels (50 microg kg(-1) for propoxur and 60 microg kg(-1) for phoxim). These results provide the first evidence that propoxur and phoxim do not penetrate from eggshell into whole egg under experimental and field conditions. Subsequently, residue carry-over after egg cracking in households and during a worst-case situation in an egg-cracking plant was investigated. However, when eggs were cracked manually, a negligible contamination of whole egg values occurred. If, in an automated process, eggshells accidentally come into close contact with whole egg, very high residue levels of propoxur and phoxim may be generated time dependently. These results suggest that eggshell contact with whole egg during egg cracking must be avoided to prevent pesticide carry-over.

Multiple effects of pentyl-4-yn-VPA enantiomers: from toxicity to short-term memory enhancement.

2-n-Pentyl-4-pentynoic acid (PE-4-yn-VPA) is a derivative of the antiepileptic and mood-stabilizing drug valproic acid (VPA). PE-4-yn-VPA exists as R- and S-enantiomers, the latter being more teratogenic. PE-4-yn-VPA also possesses antiepileptic, antiproliferative, and cell-differentiating properties. Moreover, the less teratogenic enantiomer, R-PE-4-yn-VPA, was recently shown to improve learning and memory. We here present a detailed investigation of the enantioselective properties of PE-4-yn-VPA using a range of in vitro and in vivo assays including measurements of cellular growth and migration, neuronal differentiation and survival, intracellular signal transduction, synaptic plasticity and maturation, and short-term memory as determined by the social recognition test. The results show that the enantiomers of PE-4-yn-VPA largely had similar effects in vitro. However, in all in vitro experiments the more teratogenic enantiomer, S-PE-4-yn-VPA, exhibited a stronger potency than R-PE-4-yn-VPA, and only S-PE-4-yn-VPA had a detrimental effect on cell survival. Interestingly, both the R- and S-enantiomer improved learning and memory. In contrast, the beneficial effect of S-PE-4-yn-VPA on memory was lost by time, whereas the effect of R-PE-4-yn-VPA administration was longer lasting, suggesting that the beneficial effect of the S-enantiomer on memory formation may be counteracted by its detrimental effect on neuronal cell survival.

Induction of the homeotic gene Hoxa1 through valproic acid's teratogenic mechanism of action.

BACKGROUND: Valproic acid (VPA) exposure in utero has been associated with an increased risk of both neural tube defects and autism spectrum disorders (ASDs). The terata induced by VPA suggest interference with pattern formation. Retinoic acid produces similar terata and is known to act in part by increasing the expression of Hoxa1. We tested the hypotheses that exposure to VPA would alter the expression of Hoxa1 in rat embryos during times of normal Hoxa1 expression (d10.5-13.5) and that exposure at earlier and later stages would induce inappropriate expression. METHOD: Hoxa1 expression levels were determined by real-time PCR in individual embryos 1 h after exposure on gestational d10, 12, 13, 14, or 15. Additionally, teratogenic (4-yn-VPA) and nonteratogenic analogs of VPA (IE-VPA), retinoic acid (RA), and saline were compared for effects on Hoxa1 expression on d12. Embryos were allowed to develop for 1, 2, 4, 6, or 24 h, to follow the time course of effects. RESULTS: In utero exposure to VPA on gestational d10 and on d12-14 significantly increased the level of Hoxa1 expression compared to saline-exposed embryos at developmental ages prior to, during and after the normal expression period for this gene. On gestational d12, exposures to VPA and 4-yn-VPA significantly increased Hoxa1 expression at all sacrifice times, compared to saline-exposed embryos. RA significantly elevated Hoxa1 expression at all time points except 24-h post-treatment. The nonteratogenic VPA analog, IE-VPA, did not affect Hoxa1 expression. CONCLUSIONS: VPA and 4-yn-VPA exposures elevated Hoxa1 mRNA during its normal expression period and induced expression outside of the normal period. This may explain, in part, how VPA disrupts development.

Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding protein.

Using Rbp4-null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor gamma (RARgamma) in spermatogonia and by RARalpha in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene.

Novel valproic acid derivatives with potent differentiation-inducing activity in myeloid leukemia cells.

The anti-epileptic drug valproic acid harbors anti-tumoral activity in solid and leukemic tumor cell models and is currently evaluated in clinical trials. However, the plasma trough concentrations obtained in patients by common anti-epileptic dose regimens are below concentrations required for exerting anti-tumor effects in vitro. Here, we describe the identification of three novel valproic acid derivatives with superior differentiation-inducing and anti-proliferative activities in K562 bcr/abl-positive chronic myeloid leukemia cells and HL60 promyelocytic leukemia cells at achievable therapeutic VPA concentrations. These compounds reveal potent inhibition of histone deacetylase activity, induction of p21Cip/Waf expression as well as low toxicity on CD34+ bone marrow cells.

Analysis of reproductive toxicity and classification of glufosinate-ammonium.

CONCLUSION REGARDING CLASSIFICATION OF GLUFOSINATE-AMMONIUM: Science Partners' Evaluation Group (Evaluation Group) has conducted an independent analysis of the herbicide glufosinate-ammonium (GA) relative to its potential to cause reproductive toxicity in humans. Further, the Evaluation Group has evaluated the implementation of Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC) and Council Directive 91/414/EEC, with respect to classification of chemicals posing potential reproductive hazards. After consideration of all information available to us relevant to the potential of glufosinate-ammonium (GA) to cause reproductive toxicity, the Science Partners Evaluation Group concludes that no classification of GA is justified. The following form the basis of this conclusion. There are no human data to suggest that GA causes reproductive toxicity in women or in their conceptus. The issue concerning possible reproductive hazard to humans is raised solely on the basis of positive animal test results that show GA to cause preimplantation or implantation losses in rats. SPECIFICALLY: a. Daily treatment with GA had no detectable effect on the earliest stages of the reproductive sequence including gametogenesis, ovulation, mating and conception; b. Treatment with GA interfered with rat gestation before and at the stage when the conceptus implants into the uterus. This effect occurred at doses of 360 ppm in the feed (corresponding to daily doses of 27.8 mg/kg bw) and above; and c. After implantation, no further effect of GA on prenatal and post-natal development was recognized. Previous concerns that GA might be toxic to embryonic stages after implantation were not supported by the data. Abortions and stillbirth seen were associated with, and regarded as secondary to, maternal toxicity. There was no evidence suggesting the induction of malformations in the offspring. The mechanism underlying this adverse effect in experimental laboratory animals is identified-inhibition of glutamine synthetase. Glutamine is essential to the viability of the embryo. The embryo is dependent on a maternal source of the amino acid. For embryo lethality to occur, a significant reduction of maternal glutamine is required. Such reduction in maternal glutamine depends on a significant inhibition of glutamine synthetase by GA. This can only occur when the mother is exposed to very high levels of GA. SPECIFICALLY: a. The reproductive toxicity of GA is confined to very short, early stages of reproduction, during which the conceptus is dependent on maternal glutamine; and b. In order for the effect to occur, significant reduction in maternal blood glutamine level is required, which in turn depends on a significant inhibition of glutamine synthetase, induced by high levels of GA in the maternal system. There is no evidence for accumulation of GA in the mammalian organism beyond a factor of two and no evidence for its metabolic toxification. To raise a concern in humans, women would have to be exposed to GA during the very limited time frame of preimplantation or implantation and the exposure would have to be to the exceedingly high levels necessary to alter the maternal metabolism and, correspondingly, result in glutamine levels in maternal tissue and blood plasma being drastically reduced. There is no basis to suggest that such exposures would occur under conditions of normal handling and use. SPECIFICALLY: a. Under conditions of normal handling and use, operators would never be exposed to GA levels that could potentially inhibit glutamine synthetase to the extent that this inhibition could impair preimplantation or implantation. b. All acceptable exposure measurements and predictive calculations confirm this conclusion, and in fact demonstrate that reasonably foreseeable exposure of workers would be to levels significantly below the AOEL. c. The evidence is also clear that there is no reproductive toxicity hazard to workers upon reentry tosprayed fields, bystanders, consumers or toddlers. The safety margin compared to the NOAEL in animal studies is sufficiently large to assure protection of the health of workers using GA as well as bystanders, consumers, and toddlers. Pursuant to Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC), to justify a classification of category 2 there must be sufficient evidence to produce a strong presumption that human exposure to the substance may result in impaired fertility in humans. It is the conclusion of the Science Partners Evaluation Group that there is no reasonable evidence to suggest a strong presumption of impairment. To the contrary, there is clear evidence demonstrating a strong presumption that exposure to GA would not cause the adverse effect demonstrated in rats. Pursuant to Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC), to justify a classification of category 3, there must be sufficient evidence to provide a strong suspicion of impaired fertility in humans. There is no basis to conclude that the animal data demonstrating impaired preimplantation or implantation has any relevance to humans in that the effect found in rats only occurs at levels which would never be experienced by workers under conditions of normal handling and use or by bystanders, consumers, or toddlers.