Primary cilia respond to intermittent low-magnitude, high-frequency vibration and mediate vibration-induced effects in osteoblasts.

Our objective was to investigate the role of primary cilia in low-magnitude, high-frequency vibration (LMHFV) treatment of MC3T3-E1 osteoblasts (OBs). We used chloral hydrate (CH), which has a well-characterized function in chemically removing primary cilia, to elucidate the role of primary cilia in LMHFV-induced OB osteogenic responses through cell viability assay, Western blot analysis, real-time quantitative RT-PCR, and histochemical staining methods. We observed a significant, 30% decrease in the number of MC3T3-E1 OBs with primary cilia (reduced from 64.3 ± 5%) and an approximately 50% reduction in length of primary cilia (reduced from 3 ± 0.8 µm) after LMHFV stimulation. LMHFV stimulation upregulated protein expression of the bone matrix markers collagen 1 (COL-1), osteopontin (OPN), and osteoclacin(OCN) in MC3T3-E1 OBs, indicating that LMHFV induces osteogenesis. High-concentration or long-duration CH exposure resulted in inhibition of MC3T3-E1 OB survival. In addition, Western blot analysis and RT-PCR revealed that CH treatment prevented LMHFV-induced osteogenesis. Furthermore, decreased alkaline phosphate activity, reduced OB differentiation, mineralization, and maturation were observed in CH-pretreated and LMHFV-treated OBs. We showed that LMHFV induces morphological changes in primary cilia that may fine-tune their mechanosensitivity. In addition, we demonstrated the significant inhibition by CH of LMHFV-induced OB mineralization, maturation, and differentiation, which might reveal the critical role of primary cilia in the process.

Effect of trichloroacetaldehyde on the activation of CD4+T cells in occupational medicamentosa-like dermatitis: An in vivo and in vitro study.

Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is a hypersensitivity disease with autoimmune liver injury, which has increasingly become a serious occupational health problem in China. However, the pathogenesis of OMLDT remained undefined. In this study, 30 TCE-induced OMLDT patients, 58 exposure controls, and 40 non-exposure controls were recruited. We showed that the ratio of activated CD4+ T cells (downregulation of CD62 L) was dramatically increased in OMLDT patients compared to exposure and non-exposure control, suggesting that CD4+ T cells activation was a key cellular event in the development of OMLDT. In parallel, the expression of cytokine including IL-2, IFN-γ, TNF-α and IL-17A were increased obviously and IL-4 decreased in CD4+ T cells from OMLDT patients. in vitro assay, we found that trichloroethylene metabolites trichloroacetaldehyde (TCAH), not trichloroacetic acid (TCA) or Trichloroethanol (TCOH) could activate the naïve CD4+ T cells characterized by a rise in intracellular calcium, down-regulated CD62 L and subsequently trigger the secretion of IL-2, IFN-γ and TNF-α. Notably, the phosphorylation status of NF-κB and p38MAPK were elevated in OMLDT patients. Moreover, TCAH also could activate the p38MAPK and NF-κB, suggesting the role of p38MAPK and NF-κB pathways in the activation of CD4+ T cells. In addition, we found that the inhibition of Schiff base formation decreased the ability of TCAH to induce the activation of naïve CD4+ T cells and p38MAPK and NF-κB pathway. In conclusion, we revealed that the CD4+ T activation and increased the cytokines including IL-2, IFN-γ and TNF-α but decreased IL-4 in CD4+ T cells were associated with OMLDT. TCAH could activate naïve CD4+ T cells through NF-κB and p38MAPK activation induced by Schiff base formation, which might contribute to the development of OMLDT. These findings provide a new insight into the pathogenesis of OMLDT.

Towards optimized anesthesia protocols for stereotactic surgery in rats: Analgesic, stress and general health effects of injectable anesthetics. A comparison of a recommended complete reversal anesthesia with traditional chloral hydrate monoanesthesia.

Although injectable anesthetics are still widely used in laboratory rodents, scientific data concerning pain and distress during and after stereotactic surgery are rare. However, optimal anesthesia protocols have a high impact on the quality of the derived data. We therefore investigated the suitability of recommended injectable anesthesia with a traditionally used monoanesthesia for stereotactic surgery in view of optimization and refinement in rats. The influence of the recommended complete reversal anesthesia (MMF; 0.15mg/kg medetomidine, 2mg/kg midazolam, 0.005mg/kg fentanyl; i.m.) with or without reversal and of chloral hydrate (430mg/kg, 3.6%, i.p.) on various physiological, biochemical and behavioral parameters (before, during, after surgery) was analyzed. Isoflurane was also included in stress parameter analysis. In all groups, depth of anesthesia was sufficient for stereotactic surgery with no animal losses. MMF caused transient exophthalmos, myositis at the injection site and increased early postoperative pain scores. Reversal induced agitation, restlessness and hypothermia. Even the low concentrated chloral hydrate led to peritonitis and multifocal liver necrosis, corresponding to increased stress hormone levels and loss in body weight. Increased stress response was also exerted by isoflurane anesthesia. Pronounced systemic toxicity of chloral hydrate strongly questions its further use in rodent anesthesia. In view of undesired effects of MMF and isoflurane, thorough consideration of anesthesia protocols for particular research projects is indispensable. Reversal should be restricted to emergency situations. Our data support further refinement of the current protocols and the importance of sham operated controls.

2,2,2-Trichloroethanol lengthens the circadian period of Bmal1-driven circadian bioluminescence rhythms in U2OS cells.

2,2,2-Trichloroethanol (TCOH) is responsible for the pharmacological actions of chloral hydrate (CH), and is a major metabolite of trichloroethylene. Human exposure to TCOH is known to be increasing. Recently, it was reported that TCOH causes a significant phase delay of Per2 expression in mouse liver when injected daily over the course of several days. However, it is not clear whether TCOH directly modulates the molecular clock. In the present study we used a cell-based assay system to test this possibility. We found that the daily oscillation period of Bmal1 was lengthened to 3 h following treatment with 1.5 mM TCOH, and increased to 5 h with 3 mM TCOH treatment. However, low concentrations of TCOH had no noticeable effects. The effect of TCOH on Per2 oscillation was marginal. Interestingly, serum from rats anesthetized with CH also modulated Bmal1 period, suggesting that exposure to anesthesia should be taken into consideration for circadian rhythm studies. In summary, our study reveals a direct regulation of TCOH on molecular clock.

Effect of Laser wavelength on delivering appropriate laser burns through the opaque lens using a pattern scan laser.

BACKGROUND/AIMS: We evaluated the effects of pattern scan laser (PSL) wavelength in delivering appropriate laser burns to the retina of eyes with an opaque lens. METHODS: Sixteen shots of 2 × 2 square grids (64 laser spots) were delivered using green (532-nm), yellow (577-nm) and red (647-nm) lasers to the retinas of mice with mild cataract induced by chloral hydrate (400 mg/kg). Three eyes with clear lenses served as controls. One week after laser coagulation, the ratio of appropriate burns, defined as coagulation restricted to the outer half of the retina without retinal or choroidal hemorrhage, was investigated histologically. RESULTS: With the green laser, we confirmed only 3.0 ± 2.0 appropriate burns in eyes with an opaque lens, in contrast to 13.7 ± 4.0 effective burns in eyes with a clear lens. On the other hand, the yellow and red lasers produced 18 ± 5.2 and 13 ± 1.5 appropriate burns, respectively, in eyes with an opaque lens. CONCLUSION: Although all three PSL wavelengths successfully delivered appropriate burns restricted to the outer half of the retina in eyes with an opaque lens, the longer-wavelength yellow and red lasers were significantly more effective than the green laser. PSL may be a treatment option to accompany anti-vascular endothelial growth factor drug therapy.

A comparison of the effects of ketamine, chloral hydrate and pentobarbital sodium anesthesia on isolated rat hearts and cardiomyocytes.

OBJECTIVES: The study was intended to investigate which commonly used anesthetic in intact animals has the least effect on the function of isolated hearts and cardiomyocytes among the anesthetized animals. METHODS: The hearts of male Sprague-Dawley rats were removed after they were anesthetized with ketamine, chloral hydrate or pentobarbital sodium, respectively, or were cervically dislocated. They were mounted on a Langendorff shelf. Heart rate (HR), left ventricular systolic pressure (LVSP), and maximal rate of increase of left ventricular pressure (+dp/dt) were observed and recorded. Cell shorting amplitude and survival rate were detected in isolated cardiomyocytes. RESULTS: The application of ketamine and pentobarbital sodium led to a significant decrease in HR, LVSP and +dp/dt in isolated hearts. Furthermore, pentobarbital sodium inhibited cell shorting amplitude and reduced the survival rate of isolated cardiomyocytes. Chloral hydrate did not significantly alter HR, LVSP, +dp/dt, cell shorting amplitude and survival rate. CONCLUSION: The effects of anesthetics on cardiac parameters were considered when choosing an anesthesia administration. The results suggested that chloral hydrate as an anesthetic was appropriately applied for the studies of isolated hearts and cardiomyocytes.

Cardiac arrhythmias induced by chloral hydrate in rhesus monkeys.

Chloral hydrate has been long used as a safe sedative and hypnotic drug in humans. However, reports on its cardiovascular adverse effects have been published from time to time. The present study was undertaken to use Rhesus monkeys as a model to define the dose regiment of chloral hydrate at which cardiac arrhythmias can be induced and the consequences of the cardiac events. Male Rhesus monkeys of 2-3 years old were intravenously infused with chloral hydrate starting at 50 mg/kg with an increasing increment of 25 mg/kg until the occurrence of cardiac arrhythmias. In addition, a traditional up-and-down dosing procedure was applied to define a single dose level at which cardiac arrhythmias can be induced. The data obtained showed that when the sequentially escaladed dose reached 125 mg/kg, cardiac arrhythmias occurred in all monkeys tested. The single effective dose to cause cardiac arrhythmias calculated from the crossover analysis was 143 ± 4 mg/kg. This value would be equivalent to 68.6 ± 1.9 mg/kg for children and 46.4 ± 1.3 mg/kg for adults in humans. Under either multiple or single dose condition, cardiac arrhythmias did not occur before 40 min after the onset of anesthesia induced by chloral hydrate. Cardiac arrhythmias were recovered without help at the end of the anesthesia in most cases, but also continued after the regain of consciousness in some cases. The cardiac arrhythmias were accompanied with compromised cardiac function including suppressed fractional shortening and ejection fraction. This study thus suggests that cautions need to be taken when chloral hydrate is used above certain levels and beyond a certain period of anesthesia, and cardiac arrhythmias induced by chloral hydrate need to be closely monitored because compromised cardiac function may occur simultaneously. In addition, patients with cardiac arrhythmias induced by chloral hydrate should be monitored even after they are recovered from the anesthesia.

Chloral hydrate decreases gap junction communication in rat liver epithelial cells.

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1­5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell­cell contact was apparent in CH (0.5­5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.

DNA damage induction by two halogenated acetaldehydes, byproducts of water disinfection.

Drinking water contains disinfection byproducts, generated by the interaction of chlorine (or other disinfecting chemicals) with organic matter, anthropogenic contaminants, and bromide/iodide naturally present in most source waters. One class of these chemicals is the halogenated acetaldehydes (HAs), identified in high quantities when ozone is used as primary or secondary disinfectant. In this study, an analysis of the genotoxic potential of two HAs, namely tribromoacetaldehyde (TBA) and chloral hydrate (CH) has been conducted in human cells (TK6 cultured cells and peripheral blood lymphocytes). The comet assay was used to 1) measure the induction of single and double-strand DNA breaks, 2) evaluate the capacity of inducing oxidative DNA damage, and 3) determine the DNA repair kinetics of the induced primary genetic damage. In addition, chromosome damage, as a measure of fixed damage, was evaluated by means of the micronucleus test. The results of the comet assay show that both compounds are clearly genotoxic, inducing high levels of DNA breaks, TBA being more effective than CH. According to the comet results, both HAs produce high levels of oxidized bases, and the induced DNA damage is rapidly repaired over time. Contrarily, the results obtained in the micronucleus test, which measures the capacity of genotoxic agents to induce clastogenic and aneugenic effects, are negative for the two HAs tested, either using TK6 cells or human peripheral blood lymphocytes. This would indicate that the primary damage induced by the two HAs is not fixed as chromosome damage, possibly due to an efficient repair or the death of damaged cells, which is an important point in terms of risk assessment of DBPs exposure.

Chronic exposure to a trichloroethylene metabolite in autoimmune-prone MRL+/+ mice promotes immune modulation and alopecia.

The industrial solvent trichloroethylene (TCE) is a widespread environmental contaminant known to impact the immune system. In the present study, female MRL+/+ mice were treated for 40 weeks with trichloroacetaldehyde hydrate (TCAH), a metabolite of TCE, in the drinking water. The results were compared with the data from an earlier study in which MRL+/+ mice were exposed to TCAH for 4 weeks. Following a 40-week exposure, the mice developed skin inflammation and dose-dependent alopecia. In addition, TCAH appeared to modulate the CD4(+) T-cell subset by promoting the expression of an activated/effector (i.e., CD62L(lo)) phenotype with an increased capacity to secrete the proinflammatory cytokine interferon-gamma. However, unlike what was observed after only 4 weeks of exposure, TCAH did not significantly attenuate activation-induced cell death (AICD) or the expression of the death receptor FasL in CD4(+) T cells. Some metalloproteinases (MMPs) are thought to play a role in susceptibility to AICD by inducing FasL shedding. Thus, both the 4- and 40-week sera were tested for MMP-7 levels in an attempt to explain the disparate results of TCAH on AICD and FasL expression. Serum MMP-7 levels were significantly higher in mice exposed to TCAH for 4 weeks. In contrast, the serum MMP-7 levels were increased in all the mice by 40 weeks when compared with a nonautoimmune strain. Taken together, a chronic exposure to TCAH promotes alopecia and skin inflammation. The early effects of TCAH on MMP-7 levels may provide a mechanism by which TCAH promotes skin pathology.

Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid.

The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3-3mM CH for 3 days or 0.03-3mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3mM/day for 10 days, while in another cytotoxicity was seen at 1mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313-331] supporting the view that glutathione derived metabolites are likely to be responsible for nephrotoxicity.

Using the nucleolar biomarker and the micronucleus test on in vivo fish fin cells.

This study was aimed at developing the nucleolar biomarker and the micronucleus test on in vivo fish fin cells for assessing water cytotoxicity and genotoxicity. Both biomarkers can be used either jointly or separately on fins of the same fish during the experiment. For studying the nucleolar characteristics, small pieces of the fin edge were cut several times during 30-180 min of fish exposure. For micronucleus testing, the fin tissue regenerating after its cutting was investigated after 2-5 days of fish incubation. Effects of copper (0.1 and 2.5 mg/L), cadmium (0.005 and 1.0 mg/L) ions and chloral hydrate (400 and 800 mg/L) solutions were studied on cells of common carp (Cyprinus carpio L.), crucian carp (Carassius auratus gibelio Bloch.), and Mozambique tilapia (Tilapia (Sautherodon) mossambica) using a set of nucleolar characteristics (the number of nucleoli per cell, the size of a single nucleolus, and the percentage of cells with heteromorphic paired nucleoli) and the frequencies of cells with micronuclei and double nuclei. Substantial changes in parameters of nucleolar activity of fin cells were found to be caused by cadmium and copper impact. In comparison to blood cells, gill and fin cells were more sensitive as demonstrated by their nuclear damages after the chloral hydrate influence. Fin cells were useful to determine periodically cytotoxic and genotoxic effects of organic and inorganic substances in the same individual fish without any disruption of its physiological functions.

The effect of boiling water on disinfection by-product exposure.

Chloraminated and chlorinated waters containing bromide were used to determine the impact of boiling on disinfection by-product (DBP) concentrations. No significant changes were detected in the concentrations of the dihalogenated haloacetic acids (DXAAs) (i.e., dichloro-, bromochloro-, dibromoacetic acid) upon boiling of chloraminated water, whereas the levels of the trihalogenated haloacetic acids (TXAAs) (i.e., trichloro- (TCAA), bromodichloro- (BDCAA), dibromochloroacetic acid (DBCAA)) decreased over time (e.g., 9-37% for TCAA). Increased DXAA concentrations (58-68%) were detected in the boiled chlorinated sample, which likely resulted from residual chlorine reacting with DXAA precursors. TCAA concentration was unchanged after boiling chlorinated water for 1 min, but a 30% reduction was observed after 5 min of boiling. BDCAA concentrations decreased 57% upon boiling for 1 min and were completely removed after 2 min of boiling, whereas DBCAA was removed after boiling chlorinated water for 1 min. Trihalomethane concentrations were reduced in both chloraminated (74-98%) and chlorinated (64-98%) water upon boiling. Boiling chloraminated water for 1 min reduced chloroform concentration by 75%. Chloroform was reduced by only 34% in chlorinated water after a 1 min boil, which indicates that simultaneous formation and volatilization of chloroform was occurring. Most of the remaining DBPs (e.g. haloketones, chloral hydrate, haloacetonitriles) were removed by at least 90% after 1 min of boiling in both samples. These data suggest that other mechanisms (e.g., hydrolysis) may have been responsible for removal of the non-volatile DBPs and further highlight the importance of examining individual species when estimating thermal effects on DBP concentrations.

Exposure to trichloroethylene and its metabolites causes impairment of sperm fertilizing ability in mice.

Trichloroethylene (TCE) is a prevalent occupational and environmental contaminant that has been reported to cause a variety of toxic effects. Here, we have undertaken studies to test the hypothesis that TCE exposure adversely affects sperm function and fertilization. Sperm retrieved from mice exposed to TCE (1000 ppm) by inhalation for 1 to 6 weeks were incubated in vitro with eggs isolated from superovulated female mice. The number of sperm bound per egg was significantly decreased when mice were exposed to TCE for 2 and 6 weeks but not at exposures of 1 and 4 weeks. In vivo fertilization was also determined in superovulated female mice mated with males exposed to TCE for 2 to 6 weeks. The percentages of eggs fertilized, as assessed by the presence of two pronuclei, were significantly decreased after 2 and 6 weeks of TCE exposure. A slight but insignificant decrease was observed after 4 weeks of TCE exposure. The direct effects of TCE and its metabolites, chloral hydrate (CH) and trichloroethanol (TCOH), on in vitro sperm-egg binding were also investigated. Sperm-egg binding was significantly decreased when sperm were pretreated with CH (0.1-10 microg/mL). Significantly lower levels of sperm-egg binding were also detected with TCOH (0.1-10 microg/mL), although the decreases were not as pronounced as those for CH. These results showed that TCE exposure leads to impairment of sperm fertilizing ability, which may be attributed to TCE metabolites, CH, and TCOH.

Body weight considerations in the B6C3F1 mouse and the use of dietary control to standardize background tumor incidence in chronic bioassays.

In B6C3F1 mice, the rate of body growth influences susceptibility to liver neoplasia and large variations in body weight can complicate the interpretation of bioassay data. The relationship between body weight and liver tumor incidence was calculated for historical control populations of male and female ad libitum-fed mice (approx. 2,750 and 2,300 animals, respectively) and in populations of male and female mice which had been subjected to forced body weight reduction due to either dietary restriction or exposure to noncarcinogenic chemicals (approx. 1,600 and 1,700, respectively). Resulting tumor risk data were then used to construct idealized weight curves for male and female B6C3F1 mice; these curves predict a terminal background liver tumor incidence of 15-20%. Use of dietary control to manipulate body growth of male B6C3F1 mice to fit the idealized weight curve was evaluated in a 2-year bioassay of chloral hydrate. Cohorts of mice were successfully maintained at weights approximating their idealized target weights throughout the study. These mice exhibited less body weight variation than their ad libitum-fed counterparts (e.g., standard deviations of body weight were 1.4 and 3.4 g for respective control groups at 36 weeks). Historical control body weight and tumor risk data from the two male mouse populations were utilized to predict background liver tumor rates for each experimental group of the chloral hydrate study. The predicted background tumor rates closely matched the observed rates for both the dietary controlled and ad libitum-fed chloral hydrate control groups when each mouse was evaluated according to either its weekly food consumption or its weekly change in body weight.

Dietary controlled carcinogenicity study of chloral hydrate in male B6C3F1 mice.

Chloral hydrate, which is used as a sedative in pediatric medicine and is a by-product of water chlorination, is hepatocarcinogenic in B6C3F1 mice, a strain that can exhibit high rates of background liver tumor incidence, which are associated with increased body weight. In this study, dietary control was used to manipulate body growth in male B6C3F1 mice in a 2-year bioassay of chloral hydrate. Male B6C3F1 mice were treated with water or 25, 50, or 100 mg/kg chloral hydrate by gavage. The study compared ad libitum-fed mice with dietary controlled mice. The latter received variably restricted feed allocations to maintain their body weights on a predetermined "idealized" weight curve predictive of a terminal background liver tumor incidence of 15-20%. These mice exhibited less individual body weight variation than did their ad libitum-fed counterparts. This was associated with a decreased variation in liver to body weight ratios, which allowed the demonstration of a statistically significant dose response to chloral hydrate in the dietary controlled, but not the ad libitum-fed, test groups. Chloral hydrate increased terminally adjusted liver tumor incidence in both dietary controlled (23.4, 23.9, 29.7, and 38.6% for the four dose groups, respectively) and ad libitum-fed mice (33.4, 52.6, 50.6, and 46.2%), but a statistically significant dose response was observed only in the dietary controlled mice. This dose response positively correlated with markers of peroxisomal proliferation in the dietary controlled mice only. The study suggests that dietary control not only improves terminal survival and decreases interassay variation, but also can increase assay sensitivity by decreasing intra-assay variation.

Toxicokinetics of chloral hydrate in ad libitum-fed, dietary-controlled, and calorically restricted male B6C3F1 mice following short-term exposure.

Chloral hydrate is widely used as a sedative in pediatric medicine and is a by-product of water chlorination and a metabolic intermediate in the biotransformation of trichloroethylene. Chloral hydrate and its major metabolite, trichloroacetic acid, induce liver tumors in B6C3F1 mice, a strain that can exhibit high rates of background liver tumor incidence, which is associated with increased body weight. This report describes the influence of diet and body weight on the acute toxicity, hepatic enzyme response, and toxickinetics of chloral hydrate as part of a larger study investigating the carcinogenicity of chloral hydrate in ad libitum-fed and dietary controlled mice. Dietary control involves moderate food restriction to maintain the test animals at an idealized body weight. Mice were dosed with chloral hydrate at 0, 50, 100, 250, 500, and 1000 mg/kg daily, 5 days/week, by aqueous gavage for 2 weekly dosing cycles. Three diet groups were used: ad libitum, dietary control, and 40% caloric restriction. Both dietary control and caloric restriction slightly reduced acute toxicity of high doses of chloral hydrate and potentiated the induction of hepatic enzymes associated with peroxisome proliferation. Chloral hydrate toxicokinetics were investigated using blood samples obtained by sequential tail clipping and a microscale gas chromatography technique. It was rapidly cleared from serum within 3 h of dosing. Trichloroacetate was the major metabolite in serum in all three diet groups. Although the area under the curve values for serum trichloroacetate were slightly greater in the dietary controlled and calorically restricted groups than in the ad libitum-fed groups, this increase did not appear to completely account for the potentiation of hepatic enzyme induction by dietary restriction.

Mixture toxicity of priority pollutants at no observed effect concentrations (NOECs).

Environmental exposure situations are often characterised by a multitude of heterogeneous chemicals with ambiguous or unknown modes of action present at low concentrations. While multiple exposure is widely acknowledged, arguments are raised that adverse combined effects might not be evoked by mixtures of substances with dissimilar modes of action and being present at only low concentrations. In this study the combined effect of a multiple mixture composed of structurally dissimilar priority pollutants with mostly unknown modes of action has been investigated using an algal biotest. The concentrations of the components in the mixture equalled statistically estimated, individual no observed effect concentrations (NOECs). The observed mixture toxicity was not only clearly higher than expected for any single substance alone, but also well predictable using the concept of independent action.