Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB.

The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

Peripheral neuropathy in myotonic dystrophy. Relation to glucose intolerance.

Motor nerve conduction velocity (MNCV) was measured in three peripheral nerves in each of 24 patients with myotonic dystrophy having various degress of associated glucose intolerance and was compared with results in a control group. Both groups had similar glucose tolerance and were matched for age and adiposity. The mean MNCV in the ulnar, median, and peroneal nerves in the myotonic dystrophy group were all significantly slowed (P less than .01). This peripheral nerve dysfunction, usually subclinical, is yet another manifestation of this multisystem disease and is unrelated to the associated glucose intolerance.

Mechanistic considerations for carcinogenic risk estimation: chloroform.

Chloroform has been reported to induce cancer in rodents after chronic administration of high doses by gavage. However, the interpretation of these findings is hampered by a lack of knowledge concerning the relative roles of genetic and nongenetic mechanisms in these bioassays. The present studies were carried out in male B6C3F1 mice in order to investigate the potential of chloroform to induce genetic damage and/or organ toxicity at the sites where tumors have been observed in the various bioassays. These studies revealed that carcinogenic doses of chloroform produced severe necrosis at the sites where tumors later developed. This was demonstrated by light microscopy as well as by determination of the cellular regeneration index following administration of 3H-thymidine. Noncarcinogenic doses of chloroform failed to induce these responses. In contrast, studies of DNA alkylation and DNA repair in vivo failed to give any indication that chloroform had produced the type of genetic alterations associated with known genotoxic chemicals. These data suggest that the primary mechanism of chloroform-induced carcinogenesis is nongenetic in nature. If the same mechanism predominates in man, there should be little to no carcinogenic risk associated with exposure to noncytotoxic levels of chloroform.

Interim results of two-year toxicological studies in rats of vinylidene chloride incorporated in the drinking water or administered by repeated inhalation.

Male and female Sprague-Dawley rats were exposed to vinylidene chloride (VDC) orally or by inhalation in 2-year toxicological studies. Interim results are included in this report. VDC was given in the drinking water at mean +/- S.D. concentrations of 0, 68 +/- 13, 106 +/- 22, and 220 +/- 35 ppm which produced mean +/- S.D. dosage levels of 0, 5.9 +/- 0.6, 10.0 +/- 1.2, and 19.3 +/- 2.7 mg/kg for male rats and 0, 7.5 +/- 0.4, 12.6 +/- 1.1, and 25.6 +/- 2.4 mg/kg for female rats. Forty-eight rats/sex/VDC level and 80 rats/sex in the control group were used in the 2-year study with an interim kill of an additional 10 rats/sex/level at 90 days. In the inhalation study, rats were exposed to 0, 10, or 40 ppm of VDC vapor 6 hr/day, 5 days/week for 5 weeks, after which the exposure levels were changed to 0, 25, and 75 ppm of VDC. Exposure continued for a total of 18 months and the rats held for observation an additional 6 months. Interim kills occurred at 1, 6 and 12 months. A separate 90-day study using 20 rats/sex/level was conducted at 0, 25, and 75 ppm of VDC vapor. There were 86 rats/sex/level in the 2-year portion of the study. The parameters monitored were: body weight, food and water consumption (drinking water study only), hematology, clinical chemistries, cytogentics of bone marrow cells (inhalation study only), mortality, terminal organ weights, and gross and histopathology. Based on interim kills and gross pathologic observations, the main conclusions are: increased cytoplasmic vacuolation of hepatocytes was seen in the livers of rats given 200 ppm VDC in drinking water or 25 or 75 ppm VDC vapor by inhalation; based on gross tumor count, tumor incidence in VDC-exposed rats was not greater than controls.

Chronic toxicity and reproduction studies of hexachlorobutadiene in rats.

Hexachlorobutadiene (HCBD), while not produced commercially in the United States, may be encountered as an unwanted by-product of certain processes associated with the chlorination of hydrocarbons. Studies were conducted to assess the potential long-term toxicity of HCBD. In a reproduction study conducted in rats, dose levels of 20 or 2.0 mg/kg-day of HCBD induced slight maternal toxicity (primarily of the kidney) but caused no adverse effects on reproductive parameters-percent pregnancy and neonatal survival/development. A decreased neonatal body weight was noted at the highest dose level of 20 mg/kg-day of HCBD. No toxicologic effects were observed among the adults at a dose level of 0.2 mg/kg-day or among the neonates at dose levels of 0.2 or 2.0 mg/kg-day of HCBD. In a chronic toxicity study in rats, ingestion of 20 mg/kg-day for up to 2 years caused multiple toxicologic effects, primarily of the kidney, including the development of renal tubular adenomas and adenocarcinomas. Ingestion of the intermediate dose level of 2 mg/kg-day caused lesser degrees of toxicity, but no evidence of neoplasia. Ingestion of the lowest dose level of 0.2 mg/kg-day of HCBD caused no effects that could be attributed to treatment. These data indicate a dose-response relationship for HCBD-induced toxicity affecting primarily the kidney. HCBD-induced neoplasms occurred only at a dose level higher than that causing discernible renal injury.

Evaluation of spinosad in a two-generation dietary reproduction study using Sprague-Dawley rats.

Spinosad, an insecticide derived from a naturally occurring bacterium via fermentation, represents a new class of insecticides acting by a novel mode of action. A dietary study was conducted in Sprague-Dawley rats in which groups of 30 rats/sex/dosage level were given diets that provided 0, 3, 10, or 100 mg spinosad/kg body weight/day, 7 days/week, for 2 successive generations. Following 10 weeks of dietary exposure, the P1 generation was mated twice to produce F1a and F1b litters. After weaning, groups of 30 rats/sex/dosage level were selected from the F1a litters, given diets containing spinosad for 12 weeks, and mated to produce the F2 generation. Dietary administration of spinosad to rats at a dosage of 100 mg/kg/day over 2 generations produced parental toxicity and effects on the offspring. Among adult males, body weights and weight gains were decreased 2-9% relative to controls, with P1 males more affected than P2. Absolute and relative liver, kidney, heart, spleen, and thyroid weights were increased by from 12% to as much as 240% of control values. Histologic changes consistent with cationic amphiphilic compounds were noted in the kidneys, lungs, mesenteric lymph nodes, spleen, and thyroid of P1 and P2 males and females. In females given 100 mg/kg/day, though premating body weights were not affected, weight gains during the F1a and F1b gestation periods were depressed 15-16%. Increased incidences of dystocia, and vaginal bleeding and mortality occurred during parturition and lactation at 100 mg/kg/day. Effects on the offspring (decreased litter size and survival through day 4 of lactation) were limited to the high-dosage group. Signs indicative of poor maternal care noted in the pups (stomachs void of milk, cold, thin, etc.) were observed at 100 mg/kg/day. Early postnatal effects on the offspring were considered likely secondary to the effects in maternal animals around the time of parturition. At 100 mg/kg/day, weight gain in pups was depressed throughout lactation, with statistically significantly decreased weights noted toward the latter half of the lactation period. There were no treatment-related effects on adults or their offspring at 3 or 10 mg/kg/day in either generation. Based on these results, spinosad is not considered a selective reproductive toxicant, (i.e., no effects on reproductive parameters were noted below a level that produced toxicity in the adults) and the no observed effect level (NOEL) for both parental and reproductive/perinatal toxicity was 10 mg/kg/day.

Biofeedback training to overcome poststroke foot-drop.

The technique has exciting potential for elderly hemiplegic patients, even those who are confined to a nursing home. There are limiting factors, of course; the method is time-consuming and the initial outlay for equipment is high. But the rewards can be well worth the time, effort, and cost involved. The recovery potential of some geriatric patients after a cerebrovascular accident may seem bleak because of multiple disabilities, e.g., paralysis, delayed reflexes, aphasia. Fortunately, these disorders do not necessarily decree failure of biofeedback training. Neither does advanced age. The strongest component in success is motivation. A case in point is the 82 year old woman described here who had been hemiparetic for seven years. With biofeedback training, she gained--and maintained--muscle strength.

Effects of continuous intravenous infusion of Escherichia coli endotoxin into swine.

Escherichia coli endotoxin was continuously infused IV into 31 pigs. The response was related to the infusion rate. Of 13 pigs given endotoxin at the rate of 15.4 and 23.1 micrograms/kg of body weight/hour, 10 had bilateral renal cortical necrosis similar to a generalized Shwartzman lesion. All 13 pigs given endotoxin at the rate of 6.6 to 12.1 micrograms/kg/hour survived the infusion and were euthanatized 7 to 12 days later. Most of the pigs that developed chronic neurologic disturbances were given the smaller dose of endotoxin. Of 5 pigs given endotoxin at the rate of 22.0 to 23.1 micrograms/kg/hour after receiving 200 U of sodium heparin/kg of body weight, 1 died during the 48-hour period of infusion and the 4 pigs euthanatized 8 to 12 days later had lesions that were similar to those produced by endotoxin without heparin. The 4 control pigs given continuous IV infusion of isotonic saline solution at a rate of 30 dl/hour had no clinical signs or pathologic lesions. The continuous IV infusion of endotoxin into pigs at various doses produced similar clinical signs of endotoxic shock. Pigs that died acutely had widespread thrombosis. The hematologic findings indicated an intravascular coagulopathy and some of the clinical signs and lesions in the pigs resembled changes in the CNS in pigs with spontaneous edema disease (ED); however, distinct differences were also observed. Fibrinoid vascular necrosis did not occur in the CNS, and this lesion is consistently present in naturally occurring ED. The exact nature of the causal substance or substances causing ED is still unknown, but is considered an angiotoxin associated with hemolytic E coli.

1,1,1-trichloroethane formulation: a chronic inhalation toxicity and oncogenicity study in Fischer 344 rats and B6c3F1 mice.

Groups of male and female Fischer 344 rats and B6C3F1 mice (80/sex/group) were exposed to vapor concentrations of 0, 150, 500, or 1500 ppm 1,1,1-trichloroethane formulation 6 hr/day, 5 days/week, for 2 years. Ten rats and mice/sex from each group were predesignated for interim sacrifices after 6, 12, and 18 months of exposure. Fifty rats and mice/sex/group were assigned to the study to be terminated after 24 months. Parameters measured during the study included mortality, in-life clinical signs of toxicity, hematology, urinalysis (rats only), clinical chemistry, body weight, organ weights (liver, kidneys, brain, heart, testes), gross pathology, and histopathology. Inhalation exposure of male and female Fischer 344 rats to 1500 ppm vapor of the 1,1,1-trichloroethane formulation for 2 years resulted in a significant decrease in body weights of females. In addition, very slight microscopic hepatic effects were seen in the liver of 1500 ppm-exposed male and female rats necropsied at 6, 12, and 18 months. The hepatic effects could not be discerned at 24 months due to confounding geriatric changes. In the rats exposed to 150 and 500 ppm there were no changes that were considered due to exposure to the 1,1,1-trichloroethane formulation. There were no toxic effects noted in male or female mice at any exposure concentration tested. There were no indications of an oncogenic effect in rats or mice following 2 years of exposure to this 1,1,1-trichloroethane formulation.

Teratologic evaluation of 2-phenoxyethanol in New Zealand White rabbits following dermal exposure.

2-Phenoxyethanol was applied to the clipped skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol, and fetuses were examined for external, visceral, and skeletal alterations. Dermal application of 1000 mg/kg/day produced maternal toxicity as evidenced by intravascular hemolysis of red blood cells and death in some animals. Maternal toxicity was observed in rabbits treated with 600 mg 2-phenoxyethanol/kg/day but at a lower incidence than that observed at 1000 mg/kg/day. Nine rabbits in the 1000 mg/kg/day dose group and five rabbits at 600 mg/kg/day died or were sacrificed in extremis. Rabbits in the two highest dose groups which survived until Day 28 of gestation showed no evidence of treatment-related effects. No signs of maternal toxicity were seen at 300 mg/kg/day. Examination of rabbit fetuses indicated that, at the dosages tested, 2-phenoxyethanol was not embryotoxic, fetotoxic, or teratogenic.

Effects of teatcup liner tension on teat canal keratin and teat condition in cows.

The effect of tension of teatcup liners on teat end condition and quantity of keratin in the teat canal was investigated. Liner tension was increased by using longer teatcup shells. The first experiment used six Holstein cows in early lactation. Left quarters were milked with liners under medium or normal tension by using Conewango liners in 142 mm shells. Right quarters were milked with liners under high tension by mounting the liners in teatcup shells 149 mm in length. By day 16 teat end condition and sensitivity to manipulation were worsened by thrice daily milking when liners were under a higher tension. Two subsequent experiments each used 12 different Holstein cows. These cows were in mid lactation and were milked twice daily for 10 or 30 d. Left quarters were milked with liners under high tension Right quarters were milked with liners under low tension by using teatcup shells 126 mm in length. The quantity of keratin removed during milking was not influenced by liner tension: however, the quantity of keratin at the end of the experiments was increased 10-20% in teats that were milked using liners under higher tension. Histological analysis and keratin content were consistent with epithelial hyperplasia induced by milking with liners under increased tension.

Evaluation of renal function in rhesus monkeys and comparison to beagle dogs following oral administration of the organic acid triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid).

The current study evaluated the effects of triclopyr (3,5, 6-trichloro-2-pyridinyloxyacetic acid) on renal function following oral administration in the beagle dog and rhesus monkey. Male rhesus monkeys were orally administered triclopyr by gavage at a dose of 5 mg/kg/day, 7 days/week for 28 days, after which the dosage was increased to 20 mg/kg/day for 102 consecutive days. Groups of male dogs were administered either a single oral dose of 5 mg/kg triclopyr or were fed a diet spiked with triclopyr at a dose of 5 mg/kg/day for 47 consecutive days. The following functional and clinical chemistry parameters were evaluated: exogenous phenolsulfonphthalein (PSP) excretion, inulin and para-aminohippurate (PAH) clearance (monkeys only), endogenous serum creatinine, and blood urea nitrogen (BUN) at multiple time points during the study. Creatinine, BUN, and inulin clearance were within the normal range from both species following triclopyr administration which indicates that repeated administration of triclopyr in the dog and monkey had no effect on glomerular filtration rate (GFR). In monkeys, the percentage excretion of PSP and PAH appeared to increase following triclopyr administration (20 mg/kg/day), suggesting that these weak organic acids may be competing for the same plasma protein-binding site enhancing their clearance. More importantly, these data strongly suggest that triclopyr is not competing with PSP or PAH for the active secretory site within the monkey kidney proximal tubules. In contrast, PSP clearance studies in dogs clearly demonstrated that triclopyr administration (5 mg/kg) can significantly decrease the percentage PSP excretion even following a single dose administration. The decrease in percentage PSP was reversible and inversely related to the plasma triclopyr concentration. Overall, these data clearly indicate that triclopyr effectively competes with PSP for the active secretory site within the dog kidney proximal tubules. In contrast, the monkey was insensitive to the effects of triclopyr on the active secretory process even at doses fourfold higher (20 mg/kg/day) than the effective dose in the dog (5 mg/kg/day). These findings suggest that the effect observed on PSP and PAH excretion in the dog represent a physiological competition for excretion and not toxicity.

Neurotoxicity of methyl chloride in continuously versus intermittently exposed female C57BL/6 mice.

This study evaluated the relationship between methyl chloride (MeCl) exposure duration and neurotoxicity. Female C57BL/6 mice were exposed to MeCl for 11 days, either continuously (22 hr/day) to 15, 50, 100, 150, or 200 ppm, or intermittently (5.5 hr/day) to 150, 400, 800, 1600, or 2400 ppm. This strain and sex of mouse was chosen because it is sensitive to MeCl neurotoxicity and was a good candidate to allow the evaluation of morphological effects and the quantitation of functional effects. A simple quantitative relationship between neurotoxicity and continuous vs intermittent exposure was not observed. Although the no-observable-effect levels for continuous and intermittent MeCl exposures were very nearly proportionate to exposure concentration multiplied by duration, the dose-response curve was much steeper for continuously exposed mice. Cerebellar granular cell layer degeneration was observed in mice exposed continuously to 100 ppm MeCl and in mice exposed intermittently to 400 ppm. This histopathologic effect was observed at lower concentrations than a decrement in rotating rod running performance. No effects were observed in mice exposed to 50 ppm continuously or to 150 ppm intermittently. Continuous exposure to MeCl produced the cerebellar lesion with less effect on other tissues than did intermittent exposure. In mice exposed to 2400 ppm intermittently, there were renal and hematopoietic effects in addition to relatively slight cerebellar granular cell layer degeneration. These 2400-ppm exposed mice developed hemoglobinuria, apparently as a result of intravascular hemolysis. Although the effect of exposure duration on MeCl toxicity was complex, this study indicated that careful judgment is necessary when extrapolating intermittent exposure data to a continuous exposure situation.

Postural effects on intra-abdominal pressure during Valsalva maneuver.

When lifting heavy loads, trunk muscle contraction converts the abdominal and thoracic cavities into a nearly rigid-walled cylinder that provides increased extrinsic stability and allows partial transfer of load away from the spine. Because twisting is a common mechanism of low-back injuries, this study was undertaken to determine if trunk rotation results in a decrease in the extrinsic stability of the spine. We studied the effects of changes in trunk posture on intra-abdominal pressure generated during a maximum effort Valsalva's maneuver (IAP max) in eight healthy volunteers. IAP max during standing combined with trunk rotation was found to be significantly lower than IAP max during standing straight (p < 0.05). IAP max during forward flexion combined with trunk rotation was significantly lower than during forward flexion (p < 0.01). The results of our study indicate that trunk rotation adversely effects the ability to perform a Valsalva's maneuver. We conclude that extrinsic stability of the spine is at a biomechanical disadvantage when the trunk is rotated and thus may be a contributing factor to twisting-type back injuries.

Neurotoxicologic examination of rats dermally exposed to 2,4-D amine for three weeks.

There was no evidence of peripheral neuropathy or other neurotoxicity in rats dermally treated with a 12% aqueous solution of the amine salt of 2,4-dichlorophenoxyacetic acid (2,4-D amine). Male and female Fischer 344 rats were treated on the skin of all four limbs with 2,4-D amine for 2 hr/day, 5 days/week, for 3 weeks. Measurements were: body weights, hindlimb grip strength, accelerating rod performance, single and paired pulse electrophysiology of the caudal and sciatic nerves, hindfoot H-reflexes, light microscopy of brain, spinal cord, sciatic nerve, tibial nerve, digital nerve, and electron microscopy of the tibial nerve. The experiment continued for up to one month postexposure. Treatment caused a weight loss in both male and female rats and caused minor skin changes during treatment in both sexes. No other treatment-related effects were found.

Teratologic evaluation of dermally applied diethylene glycol monomethyl ether in rabbits.

Diethylene glycol monomethyl ether (DEGME) was applied to the skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 50, 250, or 750 mg/kg/day of DEGME, and fetuses were examined for external, visceral, and skeletal alterations. Topical application of the highest dose, 750 mg/kg/day, produced slight embryotoxicity, fetotoxicity, and toxicity in the maternal animal. Maternal effects were characterized by decreased weight gain and a concurrent physiologic decrease in red blood cells and packed cell volume values. In addition, a slight increase in embryonic resorptions was observed. The fetal alterations observed, mild forelimb flexure, slight-to-moderate dilation of the renal pelvis, retrocaval ureter, cervical spurs, and delayed ossification of the skull and sternebral bones, are considered to be indicative of fetotoxicity but not teratogenicity. Slight fetotoxicity in the form of delayed ossification of the skull and cervical spurs was seen in the 250 mg/kg/day dose group. No adverse maternal, embryonic, or fetal effects were observed at 50 mg/kg/day.