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.

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.

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.

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.

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.

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.

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.

Tumorigenicity of chloral hydrate, trichloroacetic acid, trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal, crotonaldehyde, and acrolein in the B6C3F(1) neonatal mouse.

The tumorigenicity of chloral hydrate (CH), trichloroacetic acid (TCA), trichloroethanol (TCE), malondialdehyde (MDA), crotonaldehyde, acrolein, and 4-hydroxy-2-nonenal (HNE) was tested in the B6C3F(1) neonatal mouse. Mice were administered i.p. injections of CH (1000, 2000, 2500, and 5000 nmol per animal), TCA (1000 and 2000 nmol), TCE (1000 and 2000 nmol), MDA (1500 and 3000 nmol), crotonaldehyde (1500 and 3000 nmol), acrolein (75 and 150 nmol), and HNE (750 and 1500 nmol) at 8 and 15 days of age. At 12 months, only male mice treated with the positive control chemicals, 4-aminobiphenyl (500 and 1000 nmol) and benzo[a]pyrene (150 and 300 nmol), had incidences of tumors in the liver significantly higher than the solvent control. Additional male mice were dosed as described above and their livers were excised at 24, 48 h, and 7 days after the final dose. Liver DNA was isolated and analyzed by 32P-postlabeling/high-performance liquid chromatography (HPLC) and HPLC/electrochemical detection for MDA-derived adduct (M(1)G) and 8-oxo-2'-deoxyguanosine (8-OHdG) formation, respectively. At 24 and 48 h after the final dose, CH- and TCA-treated mice exhibited significantly higher M(1)G levels than the controls. 8-OHdG formation was also induced by CH, TCA, and MDA. These results suggest that under these experimental conditions the B6C3F(1) neonatal mouse is not sensitive to carcinogens that induce an increase in endogenous DNA adduct formation through lipid peroxidation or oxidative stress.

Toxicology of chloral hydrate in the mouse.

Chloral hydrate has been found in our drinking water supplies at levels up to 5 micrograms/1. The purpose of this study was to evalute the acute and subchronic toxicology of chloral hydrate in the random-bred CD-1 mouse, to provide data for risk assessment. The acute oral LD50 of this compound was 1442 and 1265 mg/kg in male and female mice, respectively. Acute toxicity appeared to be related to depression of the central nervous system. Fourteen-day exposure by gavage in male mice at doses 1/10 and 1/100 the LD50 caused an increase in liver weight and a decrease in spleen weight at the highest dose level. Based on the data derived from 14 days of exposure, a 90-day study was performed. The compound was delivered via the drinking water; levels of the compound delivered per day were equivalent to those dosed in the 14-day study. The target organ in both sexes appeared to be the liver, with the males most affected. Male mice demonstrated a dose-related hepatomegaly accompanied by significant changes in serum chemistries and hepatic microsomal parameters. The females did not demonstrate the hepatomegaly observed in males, but did show alterations in hepatic microsomal parameters. No other significant toxicological changes were observed in either sex following 90 days of exposure.

Humoral and cell-mediated immune status in mice exposed to chloral hydrate.

Chloral hydrate has been found in our drinking water supplies at levels up to 5 micrograms/1. The purpose of this study was to evaluate the functional status of the immune system in random-bred CD-1 mice exposed to chloral hydrate for 14 and 90 days. Male mice, following 14 or 90 days of exposure to 1/10 and 1/100 the actual oral LD50, exhibited no alterations in either humoral or cell-mediated immunity. However, female mice exposed for 90 days to chloral hydrate in the drinking water demonstrated a significant depression in humoral immune function. This depression was observed when spleen cells from exposed mice were evaluated for their ability to produce antibody against sheep erythrocytes. These females did not demonstrate any changes in cell-mediated immune status.

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.

Investigation of aneuploidy induction in mouse oocytes following exposure to vinblastine-sulfate, pyrimethamine, diethylstilbestrol diphosphate, or chloral hydrate.

The various causative and mechanistic phenomena associated with aneuploidy induction require considerable investigation to better understand the etiology of chromosome missegregation. We investigated the potential of vinblastine sulfate, pyrimethamine, diethylstilbestrol diphosphate, and chloral hydrate to induce numerical and structural chromosome changes in female mouse germ cells. Superovulated ICR mice were administered the compounds either by intraperitoneal injection or oral gavage, and oocytes were collected and processed for cytogenetic analysis 17 hr later. Vinblastine sulfate, administered i.p., induced a significant increase in the frequency of ovulated MI oocytes and of hyperploid MII oocytes compared to controls, but did not increase the frequency of structural aberrations. Pyrimethamine, diethylstilbestrol diphosphate, and chloral hydrate did not increase the frequency of numerical or structural chromosome changes in female mouse germ cells.

Micronuclei induced in round spermatids of mice after stem-cell treatment with chloral hydrate: evaluations with centromeric DNA probes and kinetochore antibodies.

The chromosomal effects of chloral hydrate (CH) on germ cells of male mice were investigated using two methods to detect and characterize spermatid micronuclei (SMN); (a) anti-kinetochore immunofluorescence (SMN-CREST) and (b) multicolor fluorescence in situ hybridization with DNA probes for centromeric DNA and repetitive sequences on chromosome X (SMN-FISH). B6C3F1 mice received single intraperitoneal (i.p.) injections of 82.7, 165.4, or 413.5 mg/kg and round spermatids were sampled at three time intervals representing cells treated in late meiosis, early meiosis, or as spermatogonial stem cells. No increases in the frequencies of SMN were detected for cells treated during meiosis using either SMN-CREST or SMN-FISH methods. After spermatogonial stem-cell treatment, however, elevated frequencies of SMN were detected by both methods. With SMN-FISH, dose trends were observed both in the frequencies of spermatids containing micronuclei and in the frequency of spermatids carrying centromeric label. These findings corroborate the recent report by Allen and colleagues [Allen JW et al.(1994): Mutat. Res. 323:81-88] that CH treatment of spermatogenic stem cells induced SMN. Furthermore, our findings suggest that chromosomal malsegregation or loss may occur in spermatids long after CH treatment of stem cells. Further studies are needed to understand the mechanism of action of the CH effect on stem cells and to determine whether similar effects are induced in human males treated with CH.

Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes.

An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri- (TCA), di- (DCA), and mono- (MCA) chloroacetic acid and their corresponding aldehydes, tri- (chloral hydrate, CH), di- (DCAA) and mono- (CAA) chloroacetaldehyde. None of the chloroacetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4 hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. The continuous exposure of mice to 5 g/L DCA in the drinking water for 7 and 14 days did not induce appreciable hepatic DNA SB (< 10% at 14 days), although peroxisome proliferation, as evidenced by an increased cyanide-insensitive palmitoyl CoA oxidase (PCO) activity, was stimulated to 490% (7 days) and 652% (14 days) of control. Under this protocol, DENA (0.1 g/L) produced DNA damage after both 7 days (73% of control) and 14 days (57% of control). Similarly, long-term exposure of rats (30 weeks) to 2 g/L DCA in the drinking water, a level that increased PCO activity to 364% of the control value, exhibited no DNA damage. Both the chloroacetic acids and the chloroacetaldehydes were ineffective in inducing DNA SB in cultured rat and mouse hepatocytes at concentrations below those that yielded cytotoxicity. The chloroacetic acids were also ineffective in the CCRF-CEM cells. However, two of the chloroaldehydes, DCAA and CAA, did induce DNA SB in the CCRF-CEM cells at concentrations that did not decrease the cell viability after 2 hr of treatment. Prior incubation of DCAA and CAA with a rat S9 liver homogenate eliminated much of the DNA damaging activity. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other roden tissues and cultured cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypothermia and chloropent anesthesia differentially affect the flash evoked potentials of hooded rats.

Anesthetics and body temperature alterations are both known to alter parameters of sensory-evoked responses. However few studies have quantitatively assessed the contributions of hypothermia to anesthetic-induced changes. Two experiments were performed. In the first, chronically implanted rats were injected with either 0, 0.05, 0.10 or 0.20 ml Chloropent/100 g b.w., while body temperature was maintained. Flash evoked potentials recorded 30 min later showed increased latencies but only minor (not statistically significant) changes in amplitude. In the second experiment the same rats were anesthetized with 0.35 ml Chloropent/100 g b.w. and rectal temperature was systematically varied between 31 degrees C and 37 degrees C. Over the ranges of temperature and anesthetic employed, latencies increased more extensively with hypothermia than with anesthesia. P1N1 amplitude doubled when temperature was lowered to 31C, but P2N2 and N2P3 amplitudes declined over the same temperature range. Anesthetic-induced changes in peak-to-peak amplitude did not reach statistical significance when body temperature was constant. The findings suggest that previously reported alterations in evoked potentials following anesthesia may have been confounded with hypothermia.

Trichloroethylene inhibits aldehyde dehydrogenase only for aliphatic aldehydes of short chains in rats.

The effects of trichloroethylene (TCE) administration on aldehyde dehydrogenase (ALDH) and cytochrome P450 isozymes were studied in rats and compared with those of methanol. Intragastric administration of TCE to rats at 0.05 or 0.2 ml/kg for 1 week significantly inhibited ALDH activity for aliphatic aldehydes of short chains in the mitochondrial and cytosolic fractions of rat liver, respectively, but had no effect on the activity for long chain aliphatic aldehydes. ALDH activity catalyzing the metabolism of some aromatic aldehydes was even induced by TCE. Microsomal ALDH activity was not decreased by TCE treatment. A kinetic study showed that the low-Km isozyme of ALDH for propionaldehyde in mitochondrial and cytosolic fractions was inhibited by TCE treatment. Addition of TCE, trichloroethanol or trichloroacetic acid to the in vitro assay system did not affect the activity for acetaldehyde, but chloral hydrate at 0.02 mM decreased the activity by 42 and 35% in cytosol and the 700 x g supernatant, respectively. Methanol treatment, on the other hand, had no effect on any ALDH activity. Both TCE and methanol significantly induced CYP2E1 in rat liver. The combined effects of TCE on ALDH and cytochrome P450 may account for the degreasers' flush. Exposure to TCE and methanol may result in a change in the metabolism and toxicity of other chemicals.

Intraperitoneal injection of chloral hydrate causes intra-abdominal adhesions and unilateral testicular atrophy in golden Syrian hamsters.

We investigated the reason for the high mortality we had observed in hypophysectomized-orchidectomized Golden Syrian hamsters that were anesthetized with intraperitoneal (i.p.) injections of chloral hydrate (CH). Intact male Golden Syrian hamsters were injected intraperitoneally with 0.1cc/100g BW of a 35% solution of CH, a 35% solution of sodium chloride, or double-distilled water. Equal numbers of hamsters in each group were injected on the right or left side of the abdomen. Within 10 days, 35% of the CH-injected hamsters were dead or had to be euthanized. Autopsy revealed severe peritonitis and adynamic ileus. CH-injected hamsters that survived gained weight at a rate similar to that of the controls. All surviving hamsters were killed 18 days after the injections. Among the surviving CH-injected hamsters, 84.6% had intra-abdominal adhesions, 61.5% had unilateral testicular atrophy, and 53.8% had a yellowish necrotic mass in the epididymal fat pad (EFP). All the lesions occurred on the side that was injected. The atrophied testes had been rendered cryptorchid due to involvement with intra-abdominal adhesions. In the water-treated controls, there were no abnormalities; whereas, in the saline controls, 75% had a mass in the EFP. Histology of the EFP mass was similar in hamsters injected with CH or hypertonic saline and suggested a diagnosis of fat necrosis. The results suggest that the mortality, the intra-abdominal adhesions, and the unilateral cryptorchidism were caused by a single i.p. injection of CH, but the fat necrosis in the EFP was probably caused by high concentrations of salt. The results further suggest that high concentrations of CH should not be injected intraperitoneally for anesthesia in chronic studies, particularly of the male reproductive system.

Gastric mucosal injury induced by chloral hydrate.

Gastric mucosal injury by chloral hydrate (CH) was investigated in the rat. Rats were injected with CH intraperitoneally or subcutaneously. After the CH injection, gastric ulcers appeared in the glandular stomach at 6 h and the ulcer index peaked in 12 h. The minimum dosage of CH to produce ulcers was 400 mg/kg intraperitoneally and 600 mg/kg subcutaneously.