Dynamic effects of miR-20a-5p on hippocampal ripple energy after status epilepticus in rats.

To determine the dynamic effects of miR-20a-5p on hippocampal ripple energy in rats after status epilepticus (SE). A lithium pilocarpine (LiCl-PILO)-induced rat model of status epilepticus (SE) was established, and the rats were divided into the normal control (Control, CTL), epileptic control (PILO), valproic acid (VPA + PILO), miR-20a-5p overexpression lentivirus vector (miR + PILO), sponges blocking lentivirus vector (Sponges + PILO), and scramble sequence negative control (Scramble + PILO) groups (n = 6). Electroencephalograms (EEGs) were used to analyze changes in hippocampal ripple energy before and after SE. Quantitative polymerase chain reaction (q-PCR) analysis showed that miR-20a-5p levels gradually increased after miR-20a-5p overexpression lentivirus vector injection into the lateral ventricle, and the miR-20a-5p levels were significantly higher than that in CTL group on days 7 and 36 (P < 0.001). The miR-20a-5p levels decreased significantly on days 7 and 36 after blocking by sponges lentivirus vector injected into the lateral ventricle (P < 0.001). After injection of PILO, the average ripple energy expression in each group gradually increased, and reached the peak before chloral hydrate injection (compared with 1 day before SE, P < 0.05). The ripple energy in the VPA + PILO and Sponges + PILO groups was significantly lower than that in the PILO group at 60 min and 70 min after PILO injection and before chloral hydrate injection (P < 0.05), and maintained lower until 2 h after chloral hydrate injection in VPA + PILO (P < 0.05). Compared with the VPA + PILO group, the mean ripple energy of the Sponges + PILO group had no difference at all time points (P ≥ 0.05). After SE, ripple distribution of space and energy is closely related to the occurrence of epilepsy. Inhibition of miR20a-5p expression can downregulate ripple oscillation energy during seizure.

Trichloroethanol, an active metabolite of chloral hydrate, modulates tetrodotoxin-resistant Na+ channels in rat nociceptive neurons.

BACKGROUND: Chloral hydrate is a sedative-hypnotic drug widely used for relieving fear and anxiety in pediatric patients. However, mechanisms underlying the chloral hydrate-mediated analgesic action remain unexplored. Therefore, we investigated the effect of 2',2',2'-trichloroethanol (TCE), the active metabolite of chloral hydrate, on tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive sensory neurons. METHODS: The TTX-R Na+ current (INa) was recorded from acutely isolated rat trigeminal ganglion neurons using the whole-cell patch-clamp technique. RESULTS: Trichloroethanol decreased the peak amplitude of transient TTX-R INa in a concentration-dependent manner and potently inhibited persistent components of transient TTX-R INa and slow voltage-ramp-induced INa at clinically relevant concentrations. Trichloroethanol exerted multiple effects on various properties of TTX-R Na+ channels; it (1) induced a hyperpolarizing shift on the steady-state fast inactivation relationship, (2) increased use-dependent inhibition, (3) accelerated the onset of inactivation, and (4) retarded the recovery of inactivated TTX-R Na+ channels. Under current-clamp conditions, TCE increased the threshold for the generation of action potentials, as well as decreased the number of action potentials elicited by depolarizing current stimuli. CONCLUSIONS: Our findings suggest that chloral hydrate, through its active metabolite TCE, inhibits TTX-R INa and modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons. These pharmacological characteristics provide novel insights into the analgesic efficacy exerted by chloral hydrate.

Toward better understanding of chloral hydrate stability in water: Kinetics, pathways, and influencing factors.

Chloral hydrate (CH) is a disinfection byproduct commonly found in disinfected water, and once formed, CH may undergo several transformation processes in water distribution system. In order to understand its fate and occurrence in water, this study examined several factors that may affect the stability of CH in water, including pH, temperature, initial CH concentration, typical anions, and the presence of free chlorine and monochloramine. The results indicated that CH was a relatively stable compound (half-life ∼7 d for 20 µg/L) in ambient pH (7) and temperature (20 °C) conditions. However, the hydrolysis rate can be greatly facilitated by increasing pH (from 7 to 12) and temperature (from 20 to 60 °C) or decreasing initial CH concentration (from 10 mg/L to 20 µg/L). To quantify the influences of these factors on the CH hydrolysis rate constant (k, 1/h), which spans five orders of magnitude, this study developed a multivariate model that predicts literature and this study's data well (R(2) = 0.90). In contrast, the presence of chloride, nitrate, monochloramine, and free chlorine exhibited no significant impacts on the degradation of CH, while the CH loss in non-buffered waters spiked with sodium hypochlorite was driven by alkaline hydrolysis. In terms of reaction products, CH hydrolysis yielded mostly chloroform and formic acid and a few chloride, which confirmed decarburization as a dominant pathway and dehalogenation as a noticeable coexisting reaction.

Chloral hydrate, through biotransformation to dichloroacetate, inhibits maleylacetoacetate isomerase and tyrosine catabolism in humans.

BACKGROUND: Chloral hydrate (CH), a sedative and metabolite of the environmental contaminant trichloroethylene, is metabolized to trichloroacetic acid, trichloroethanol, and possibly dichloroacetate (DCA). DCA is further metabolized by glutathione transferase zeta 1 (GSTZ1), which is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme in tyrosine catabolism. DCA inhibits its own metabolism through depletion/inactivation of GSTZ1/MAAI with repeated exposure, resulting in lower plasma clearance of the drug and the accumulation of the urinary biomarker maleylacetone (MA), a metabolite of tyrosine. It is unknown if GSTZ1/MAAI may participate in the metabolism of CH or any of its metabolites and, therefore, affect tyrosine catabolism. Stable isotopes were utilized to determine the biotransformation of CH, the kinetics of its major metabolites, and the influence, if any, of GSTZ1/MAAI. METHODS: Eight healthy volunteers (ages 21-40 years) received a dose of 1 g of CH (clinical dose) or 1.5 µg/kg (environmental) for five consecutive days. Plasma and urinary samples were analyzed by gas chromatography-mass spectrometry. RESULTS: Plasma DCA (1.2-2.4 µg/mL), metabolized from CH, was measured on the fifth day of the 1 g/day CH dosage but was undetectable in plasma at environmentally relevant doses. Pharmacokinetic measurements from CH metabolites did not differ between slow and fast GSTZ1 haplotypes. Urinary MA levels increased from undetectable to 0.2-0.7 µg/g creatinine with repeated CH clinical dose exposure. Kinetic modeling of a clinical dose of 25 mg/kg DCA administered after 5 days of 1 g/day CH closely resembled DCA kinetics obtained in previously naïve individuals. CONCLUSIONS: These data indicate that the amount of DCA produced from clinically relevant doses of CH, although insufficient to alter DCA kinetics, is sufficient to inhibit MAAI and tyrosine catabolism, as evidenced by the accumulation of urinary MA.

Dechlorination of chloral hydrate is influenced by the biofilm adhesin protein LapA in Pseudomonas putida LF54.

LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.

Assimilative and co-metabolic degradation of chloral hydrate by bacteria and their bioremediation potential.

Although the bacterial degradation of chloral hydrate (CH) has been recognized for several decades, its degradation pathway by assimilation has not been demonstrated. In this paper, we report the isolation of the LF54 bacterial strain, which utilizes CH as its sole carbon and energy source. LF54 converted CH into trichloroethanol (TCAol), which was dehalogenated to dichloroethanol (DCAol), and CO(2) was detected as the end product. Another strain that we isolated, RS20, co-metabolized CH into TCAol. Our 16S rRNA gene sequencing and taxonomic analyses revealed that the LF54 and RS20 strains belong to the Pseudomonas and Arthrobacter genera, respectively. When the two strains were inoculated into soil microcosms, both degraded 0.3mM CH to undetectable levels (<0.01mM) within 5days. These results suggest that LF54 and RS20 could be used in the bioremediation of CH-contaminated environments.

Biodegradation and biotransformation of wastewater organics as precursors of disinfection byproducts in water.

Laboratory experiments were carried out to investigate wastewater organics as the precursors of disinfection byproducts (DBPs) in drinking water supply. The focus was on the change in wastewater DBP precursors during biological degradation under simulated natural conditions. The wastewater and its treated secondary effluent were characterized for DBP formation potential (DBPFP) and DBP speciation profile, including trihalomethanes, haloacetic acids, chloral hydrate, and nitrogen-containing DBPs. Several model organic compounds, including humic acid, tannic acid, glucose, starch, glycine, and bovine serum albumin (BSA), were used to represent the different types of organic pollutants in wastewater discharge. The results show that the DBPFP of wastewater decreased after biodegradation, but the remaining organic matter had a greater DBPFP yield with chlorine. Different model organics displayed different changes in DBPFP during biodegradation. The DBPFP remained largely unchanged for the glycine solution, decreased greatly for the tannic acid and BSA solutions, and increased nearly 3-fold for the glucose and starch solutions after 10d of biodegradation. Meanwhile, the DBPFP yield increased from 3 for glycine to 51µg DBP mg(-1) C for its degradation residue, and from 1 for glucose and starch to 87 and 38µg DBP mg(-1) C for their organic residues, respectively. Although biodegradation may effectively remove some DBP precursors, biotransformation during the process produces new DBP precursors in the form of soluble microbial products (SMPs). The experimental results reveal that SMPs may be an important source of wastewater-derived DBP precursors in natural waters.

Chloral hydrate intoxication in a 3-month-old child: avoidance of hemodialysis by an immediate determination of trichloroethanol.

BACKGROUND: Chloral hydrate is used worldwide as a first-line agent for procedural sedation in paediatric patients undergoing painless diagnostic investigations. Chloral hydrate overdoses in children and adults have been reported to cause various toxicities, including central nervous system, respiratory and cardiac depression with sometimes fatal outcome. PATIENT AND METHODS: A 3-month-old girl was admitted after an unintentional administration of a 10-fold dose of chloral hydrate (667 mg/kg). She showed respiratory insufficiency in need of intubation and ventilation. Gastric endoscopy revealed esophagitis and gastric ulcerations. To assess the need for hemodialysis, serum trichloroethanol (TCE) was determined using a mass spectrometric quantification after a methyl tertiary butyl ether extraction using an external standard method. The serum TCE level 6 h after administration was 89 mg/L and declined to 20 mg/L within 24 h. The child could be extubated the next day; her further course was uneventful. CONCLUSION: The repeated determination of serum TCE levels prevented a technically difficult and risky hemodialysis in this very young patient.

Kinetics of chloral hydrate and its metabolites in male human volunteers.

Chloral hydrate (CH) is a short-lived intermediate in the metabolism of trichloroethylene (TRI). TRI, CH, and two common metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA) have been shown to be hepatocarcinogenic in mice. To better understand the pharmacokinetics of these metabolites of TRI in humans, eight male volunteers, aged 24-39, were administered single doses of 500 or 1,500 mg or a series of three doses of 500 mg given at 48 h intervals, in three separate experiments. Blood and urine were collected over a 7-day period and CH, DCA, TCA, free trichloroethanol (f-TCE), and total trichloroethanol (T-TCE=trichloroethanol and trichloroethanol-glucuronide [TCE-G]) were measured. DCA was detected in blood and urine only in trace quantities (<2 microM). TCA, on the other hand, had the highest plasma concentration and the largest AUC of any metabolite. The TCA elimination curve displayed an unusual concentration-time profile that contained three distinct compartments within the 7-day follow-up period. Previous work in rats has shown that the complex elimination curve for TCA results largely from the enterohepatic circulation of TCE-G and its subsequent conversion to TCA. As a result TCA had a very long residence time and this, in turn, led to a substantial enhancement of peak concentrations following the third dose in the multiple dose experiment. Approximately 59% of the AUC of plasma TCA following CH administration is produced via the enterohepatic circulation of TCE-G. The AUC for f-TCE was found to be positively correlated with serum bilirubin concentrations. This effect was greatest in one subject that was found to have serum bilirubin concentrations at the upper limit of the normal range in all three experiments. The AUC of f-TCE in the plasma of this individual was consistently about twice that of the other seven subjects. The kinetics of the other metabolites of CH was not significantly modified in this individual. These data indicate that individuals with a more impaired capacity for glucuronidation may be very sensitive to the central nervous system depressant effects of high doses of CH, which are commonly attributed to plasma levels of f-TCE.

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.

Key issues in the modes of action and effects of trichloroethylene metabolites for liver and kidney tumorigenesis.

Trichloroethylene (TCE) exposure has been associated with increased risk of liver and kidney cancer in both laboratory animal and epidemiologic studies. The U.S. Environmental Protection Agency 2001 draft TCE risk assessment concluded that it is difficult to determine which TCE metabolites may be responsible for these effects, the key events involved in their modes of action (MOAs) , and the relevance of these MOAs to humans. In this article, which is part of a mini-monograph on key issues in the health risk assessment of TCE, we present a review of recently published scientific literature examining the effects of TCE metabolites in the context of the preceding questions. Studies of the TCE metabolites dichloroacetic acid (DCA) , trichloroacetic acid (TCA) , and chloral hydrate suggest that both DCA and TCA are involved in TCE-induced liver tumorigenesis and that many DCA effects are consistent with conditions that increase the risk of liver cancer in humans. Studies of S-(1,2-dichlorovinyl) -l-cysteine have revealed a number of different possible cell signaling effects that may be related to kidney tumorigenesis at lower concentrations than those leading to cytotoxicity. Recent studies of trichloroethanol exploring an alternative hypothesis for kidney tumorigenesis have failed to establish the formation of formate as a key event for TCE-induced kidney tumors. Overall, although MOAs and key events for TCE-induced liver and kidney tumors have yet to be definitively established, these results support the likelihood that toxicity is due to multiple metabolites through several MOAs, none of which appear to be irrelevant to humans.

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment: relative disposition of chloral hydrate to trichloroacetate and trichloroethanol.

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common groundwater contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

[Study on decomposed products of trichlorfon in process of gas chromatographic analysis by mass spectrometry].

The decomposed products of trichlorfon in gas chromatographic analysis were identified by mass spectrometry (MS). After MS interpretation, three decomposed products, trichloroacetaldehyde, dimethyl phosphite and dichlorvos were identified. The effects of gas chromatographic conditions on decomposed products of trichlorfon, e. g. injection temperature, injection mode and oven ramp, were studied. The experiments showed that all of the factors have effects on decomposed products of trichlorfon, however, the injection temperature is the key factor to cause trichlorfon being decomposed. The higher the injection temperature is, the bigger the amount of trichlorfon being decomposed. When the injection temperature was raised from 150 degrees C to 250 degrees C, the remaining trichlorfon fell from 86% to 20%. Therefore, on-cold column injection mode gas chromatography or high performance liquid chromatography was recommended for exact quantification of trace trichlorfon.

Pulmonary bioactivation of trichloroethylene to chloral hydrate: relative contributions of CYP2E1, CYP2F, and CYP2B1.

Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-dependent bioactivation to reactive metabolites. In this investigation, studies were undertaken to test the hypothesis that TCE metabolism to chloral hydrate (CH) is mediated by cytochrome P450 enzymes, including CYP2E1, CYP2F, and CYP2B1. Recombinant rat CYP2E1 catalyzed TCE metabolism to CH with greater affinity than did the recombinant P450 enzymes, rat CYP2F4, mouse CYP2F2, rat CYP2B1, and human CYP2E1. The catalytic efficiencies of recombinant rat CYP2E1 (V(max)/K(m) = 0.79) for generating CH was greater than those of recombinant CYP2F4 (V(max)/K(m) = 0.27), recombinant mouse CYP2F2 (V(max)/K(m) = 0.11), recombinant rat CYP2B1 (V(max)/K(m) = 0.07), or recombinant human CYP2E1 (V(max)/K(m) = 0.02). Decreases in lung microsomal immunoreactive CYP2E1, CYP2F2, and CYP2B1 were manifested at varying time points after TCE treatment. The loss of immunoreactive CYP2F2 occurred before the loss of immunoreactive CYP2E1 and CYP2B1. These protein decreases coincided with marked reduction of lung microsomal p-nitrophenol hydroxylation and pentoxyresorufin O-dealkylation. Rates of CH formation in the microsomal incubations were time-dependent and were incremental from 5 to 45 min. The production of CH was also determined in human lung microsomal incubations. The rates were low and were detected in only three of eight subjects. These results showed that, although CYP2E1, CYP2F, and CYP2B1 are all capable of generating CH, TCE metabolism is mediated with greater affinity by recombinant rat CYP2E1 than by recombinant CYP2F, CYP2B1, or human CYP2E1. Moreover, the rates of CH production were substantially higher in murine than in human lung.

Environmental contaminant and disinfection by-product trichloroacetaldehyde stimulates T cells in vitro.

It had been shown previously that MRL+/+ mice exposed to occupationally relevant doses of the environmental contaminant trichloroethylene in their drinking water developed lupus-like symptoms and autoimmune hepatitis in association with activation of Interferon-gamma (IFN-gamma)-producing CD4+ T cells. Since trichloroethylene must be metabolized in order to promote the T-cell activation associated with autoimmunity, the present study was initiated to determine whether the immunoregulatory effects of trichloroethylene could be mimicked by one of its major metabolites, trichloroacetaldehyde (TCAA). At concentrations ranging from 0.04 to 1 mM TCAA co-stimulated proliferation of murine T-helper type 1 (Th1) cells treated with anti-CD3 antibody or antigen in vitro. TCAA at similar concentrations also induced phenotypic alterations commensurate with activation (upregulation of CD28 and downregulation of CD62L) in both cloned memory Th1 cells, as well as naïve CD4+ T cells from MRL+/+ mice. TCAA-induced Th1 cell activation was accompanied by phoshorylation of activating transcription factor 2 (ATF-2) and c-Jun, two components of the activator protein-1 (AP-1) transcription factor. TCAA at higher concentrations was also shown to form a Schiff base on T cells, and inhibition of Schiff base formation suppressed the ability of TCAA to phosphorylate ATF-2. Taken together, these results suggest that TCAA promotes T-cell activation via stimulation of the mitogen-activated protein (MAP) kinase pathway in association with Schiff base formation on T-cell surface proteins. By demonstrating that TCAA can stimulate T-cell function directly, these results may explain how the environmental toxicant trichloroethylene promotes T-cell activation and related autoimmunity in vivo.

Metabolism and toxicity of trichloroethylene in epididymis and testis.

The widespread occupational exposure to trichloroethylene (TCE) led us to test the hypothesis that TCE causes toxicity in the male reproductive system. We also investigated mechanisms mediating the potential cytotoxic response. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for a total of 19 days. Exposure after the first week was interspersed by a "weekend." To estimate internal exposure, we measured the TCE metabolites, trichloroacetic acid (TCA) and trichloroethanol (TCOH), in urine at Days 4, 9, 14, and 19. Urinary excretion of TCOH was significantly higher than TCA; levels of TCOH and TCA significantly increased by the second and third week, respectively. Cytochrome P450 2E1 (CYP2E1), an enzyme involved in TCE metabolism, was localized in the epididymal epithelium and testicular Leydig cells, and was found at higher levels in the former than the latter. Immunoblotting confirmed that CYP2E1 protein was present in greater amounts in epididymis than in testis. p-Nitrophenol hydroxylation, a CYP2E1 catalytic activity, was also higher in the epididymis than in the testis. Chloral, a major TCE metabolite, was generated in microsomal incubations at significantly higher levels in epididymis than in testis. Antibody inhibition of CYP2E1 reduced chloral formation, which was more pronounced in epididymis than in testis. After 4 weeks of TCE exposure, damage to the epididymis was manifested as sloughing of epithelial cells. These results indicated that TCE is metabolized in the male reproductive tract, leading to adverse effects that are more severe in the epididymis than in the testis.

Binding of the active metabolite of chloral hydrate, 2,2,2-trichloroethanol, to serum albumin demonstrated using tryptophan fluorescence quenching.

Chloral hydrate, a sedative/hypnotic agent widely used in the pediatric population, is converted to the active metabolite 2,2,2-trichloroethanol (TCE) in the liver. Tryptophan fluorescence quenching has been used previously to show that halothane and chloroform bind saturably to serum albumin, and a similar approach is used here to demonstrate that TCE also binds to albumin. TCE quenches the steady-state tryptophan fluorescence of bovine serum albumin (BSA) in a concentration-dependent, saturable manner with a K(D) = 3.3 +/- 0.3 mmol/l. Unlike halothane and chloroform, however, TCE also elicits a concentration-dependent blue-shift in the fluorescence emission spectrum of BSA and human serum albumin. This indicates that TCE induces a conformational change in the protein, causing the tryptophan to experience a change in its chemical environment, thus shifting the peak of the emission spectrum. Circular dichroism spectroscopy revealed a decrease in the alpha-helical content of BSA from 65.8 +/- 0.4 to 62.9 +/- 0.6% when TCE was present at a concentration of 30 mmol/l, providing further evidence for a conformational change. There is evidence that TCE potentiates the action of ligand-gated ion channels such as the GABA(A) and 5-HT(3) receptors, and the present results suggest that anesthetic alcohols may act by binding to these proteins and inducing structural changes that may in turn alter protein function.

Toxicology and carcinogenesis study of chloral hydrate (ad libitum and dietary controlled) (CAS no. 302-17-0) in male B6C3F1 mice (gavage study).

[structure: see text] Chloral hydrate is used medically as a sedative or hypnotic and as a rubefacient in topical preparations, and it is often given to children as a sedative during dental and other medical procedures. Chloral hydrate is used as a central nervous system depressant and sedative in veterinary medicine and as a general anesthetic in cattle and horses. It is a byproduct of the chlorination of water and has been detected in plant effluent after the bleaching of softwood pulp. Chloral, the anhydrous form of chloral hydrate, is used as a synthetic intermediate in the production of insecticides and herbicides. Chloral hydrate was nominated for study by the Food and Drug Administration based upon widespread human exposure and its potential hepatotoxicity and the toxicity of related chemicals. A dietary control component was incorporated in response to concerns within the regulatory community relating to increased background neoplasm incidences in rodent strains used for toxicity testing and to the proposed use of dietary restriction to control background neoplasm incidence in rodent cancer studies. Male B6C3F1 mice (ad libitum-fed or dietary-controlled) received chloral hydrate (99% pure) by gavage for 2 years. 2-YEAR STUDY IN MALE MICE: Groups of 120 male mice received chloral hydrate in distilled water by gavage at doses of 0, 25, 50, or 100 mg/kg 5 days per week for 104 to 105 weeks. Each dose group was divided into two dietary groups of 60 mice. The ad libitum-fed mice had free access to feed, and the dietary-controlled mice received feed in measured daily amounts calculated to maintain body weight on a previously computed idealized body weight curve. Twelve mice from each diet and dose group were evaluated at 15 months. SURVIVAL, FEED CONSUMPTION, AND BODY WEIGHTS: Survival of dosed groups of ad libitum-fed and dietary-controlled mice was similar to that of the corresponding vehicle controls. When compared to the ad libitum-fed groups, dietary control significantly increased survival in the vehicle controls and 25 and 50 mg/kg groups. Mean body weights of all dosed groups were similar to those of the vehicle control groups throughout the study. The dietary-controlled mice were successfully maintained at or near their target idealized body weights. There was less individual variation in body weights in the dietary-controlled groups than in the corresponding ad libitum-fed groups. Feed consumption by 25 and 50 mg/kg ad libitum-fed mice was generally similar to that by the vehicle controls throughout the study. Feed consumption by 100 mg/kg ad libitum-fed mice was slightly less than that by the vehicle controls throughout the study. HEPATIC ENZYME ANALYSIS: Chloral hydrate did not significantly induce either lauric acid 4-hydroxylase activity or CYP4A immunoreactive protein in any of the dosed groups of ad libitum-fed mice. However, 100 mg/kg did significantly induce both lauric acid 4-hydroxylase activity and CYP4A immunoreactive protein in the dietary-controlled mice. Moreover, the induction response profile of CYP4A was similar to the increase in the incidence of liver neoplasms at 2 years in the dietary-controlled mice with the major effect occurring in the 100 mg/kg group. The serum enzymes alanine aminotransferase, amylase, aspartate aminotransferase, and lactate dehydrogenase were also assayed at 2 years. In the ad libitum-fed groups there was a significant increase in aspartate aminotransferase activity in the 50 mg/kg group. There were no other significant effects in any dosed group, but in general the dietary-controlled groups exhibited lower values than the corresponding ad libitum-fed groups. ORGAN WEIGHTS AND PATHOLOGY FINDINGS: The heart weight of ad libitum-fed male mice administered 100 mg/kg and the kidney weights of 50 and 100 mg/kg ad libitum-fed mice were significantly less than those of the vehicle controls at 2 years. The liver weights of all dosed groups of ad libitum-fed and dietary-controlled mice were greater than those of the vehicle control groups at 2 years, but the increases were not statistically significant. The incidence of hepatocellular adenoma or carcinoma (combined) in ad libitum-fed mice administered 25 mg/kg was significantly greater than that in the vehicle controls at 2 years. The incidences of hepatocellular carcinoma and of hepatocellular adenoma or carcinoma (combined) occurred with positive trends in dietary-controlled male mice at 2 years, and the incidence of hepatocellular carcinoma in 100 mg/kg dietary-controlled mice was significantly increased. CONCLUSIONS: Under the conditions used in this 2-year gavage study, there was some evidence of carcinogenic activity of chloral hydrate in male B6C3F1 mice based on increased incidences of hepatocellular adenoma or carcinoma (combined) in ad libitum-fed mice and on increased incidences of hepatocellular carcinoma in dietary-controlled mice. In the dietary-controlled mice, induction of enzymes associated with peroxisome proliferation was observed at higher doses.