Ricardo Ochoa

p53 +=¡ Hemizygous Knockout Mouse: Overview of Available Data

The performance of the p53 +/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institutes (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17β-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-defi cient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.

Correlation of dermal-epidermal laminar lesions of equine hoof with various disease conditions.

Dermal and epidermal laminar lesions were correlated with acute intestinal, primary hepatic, septicemic, chronic laminar, and acute laminar diseases. Horses with acute intestinal disease had edema in the secondary dermal laminae. Those with hepatic disease had increased keratinization of the secondary epidermal laminae. Septicemia caused increased keratin formation in the primary and secondary epidermal laminae. Chronic laminitis caused architectural changes of the epidermal laminae characterized by hyperplasia and keratin formation of the basal epidermal layer. Horses with acute laminitis had epidermal necrosis, especially with peracute laminitis. Various insults to the epidermal laminae led to epithelial hyperplasia of the secondary epidermis with ventral deviation of the third phalanx.

Adrenal cortical response in clinically normal dogs before and after adaptation to a housing environment

58 dogs (29 males and 29 females) selected as healthy on clinical and biochemical evaluations were subjected to an ACTH adrenal function test 2 days after their admission to a veterinary hospital (t+0). Basal female serum cortisol concentrations were significantly higher than concentrations in males (77 nmol/l versus 43 nmol/l; P<0·01). Concentrations post stimulation were not statistically different (P>0·05) between males and females: 306 (±69) nmol/l versus 291 (±73) nmol/l, respectively. Twelve dogs (6 males and 6 females), randomly selected from the 58, were subjected to the same test 5 weeks later (t+5) and 12 weeks later (t+12). Basal cortisol concentrations were lower at t+5 or at t+12 than at t+0. Post stimulation mean cortisol concentrations were lower in males than in females at t+5 (162 versus 232 nmol/l; P<0·05) but not at t+0 (262 versus 320 nmol/l; P>0·5) and t+12 (188 versus 233 nmol/l; P>0·05). These findings are indicating an increased susceptibility of bitches to environmental stress.

Irradiated larval vaccination of ponies against Strongylus vulgaris

Nonimmune pony foals 9 to 12 mo of age were vaccinated with third-stage Strongylus vulgaris larvae (L3) irradiated with 70, 100, or 130 Kr of gamma radiation. Ponies receiving per os inoculations of L3 irradiated with 70 or 100 Kr were protected from the clinical disease and lesions associated with challenge infections of 4,300 L3, when compared to nonvaccinated controls. Similarly, the numbers of worms from the challenging population recovered from successfully vaccinated animals were significantly lower than from nonvaccinated controls. The degree of resistance that develops in individuals can be semiquantitated based on clinical and pathological responses.

The effects of Clostridium perfringens type A enterotoxin in Shetland ponies--clinical, morphologic and clinicopathologic changes.

Severe abdominal pain, classic colic signs and hemorrhagic gastro-entero-cecocolitis were induced in three conventional Shetland ponies by intravenous injection with Clostridium perfringens Type A enterotoxin. Histological examination showed marked congestion, edema and hemorrhage of the large and small intestine and sloughing of the tips of the intestinal villi. Marked vacuolar degeneration of hepatocytes with dilatation of the spaces of Disse also was found. Clinical changes consisted of severe hypoglycemia, markedly increased aspartate aminotransferase levels and leukopenia that occurred rapidly.

Hepatic protection by 16,16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1-induced injury in the rat

Studies were conducted to assess the possible protective action of 16,16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1 (AFB1) induced hepatic injury in the rat. Evaluation of liver damage by histopathologic techniques and clinical chemistry indicated that hepatic necrosis was ameliorated by treatment with DMPG even though binding of radiolabeled (3H)-AFB1 to hepatic DNA was unaffected by this prostaglandin. However, DMPG did not protect rats against AFB1-induced mortality. These data suggest that hepatic protection by DMPG was due to mechanisms other than an interference with the activation or hepatic binding of AFB1.

Efficacy of ivermectin (22,23-dihydroavermectin B1) against adult Setaria equina and microfilariae of Onchocerca cervicalis in ponies.

number of N. brasiliensis present prior to the start of rejection on day 6 (168, 84, and 144 in Experiments 1-3, respectively) was considered 100% of the population at risk and percentages present on subsequent examination dates for each experiment were determined from that base. A delay in rejection of N. brasiliensis was apparent by day 10 in hosts with N. dubius and obviously more pronounced on days 10 and 18 in hosts with more than 100 N. dubius than in hosts with less than 30 N. dubius. The results suggest that delayed rejection of N. brasiliensis, apparent on days 10 and 18, was directly related to numbers of N. dubius, whereas prolonged survival of N. brasiliensis, apparent on days 60 and 90, was more dependent on presence than numbers of N. dubius. umber of N. brasilien is pre ent p ior to the tart of rejection on day 6 (168, 84, and 144 in The large r duction in initial development of N. brasiliensis in mice infected with N. dubius reported by Bruna and Zenia (loc. cit.) was not observed in the present work. In two of the three experiments, slight reductions in numbers of N. brasiliensis recovered on day 6 were noted, but these differences were not statistically significant. Nevertheless, this potential difference as well as the dose-dependent relationship between the two parasites observed early in the course of the N. brasiliensis infection in the present study should be noted by investigators using this model system. The investigation was supported in part by funds provided by the State of Washington Initiative Measure No. 171. e large reductio initial development of

Morphogenesis of Postmortem Hepatocyte Vacuolation and Liver Weight Increases in Sprague-Dawley Rats:

Accurate interpretation of microscopic changes in tissues is critical in hazard identification and risk assessment. To address a possible confounder, the effects of postmortem interval on hepatocyte vacuolation and liver weight were studied in fasted and nonfasted Sprague-Dawley rats. Male and female rats (5/sex/interval) were euthanized with CO2, weighed, and necropsied either immediately or after remaining in the closed CO2 chamber for 5, 10, or 25 minutes after respirations ceased. The liver was removed, weighed, and fixed for light microscopy, immunohistochemistry, and electron microscopy. The liver weight and liver to body weight ratio increased significantly in both male and female rats. Postmortem hepatocellular vacuolation was more prominent in males than in females. Both fasted and nonfasted males were similarly affected, however, fasted females were affected more than nonfasted females at the 25-minute interval. Ultrastructurally, intracytoplasmic vacuoles in hepatocytes and/or endothelial cells contained electron-lucent material that was morphologically similar to plasma in sinusoidal spaces. Results of our study suggest that hepatocyte vacuoles were formed in a postmortem time-dependent manner as a result of plasma influx into the cytoplasm. This change was associated with hepatic sinusoidal congestion and increases in liver weight. Males were more sensitive than females to postmortem hepatocyte vacuolation.

Antioxidant-dependent inhibition of diquat-induced toxicity in vivo

The abilities of two experimental antioxidants (U-74006F and U-78517G), as well as the model antioxidant, diphenyl-p-phenylenediamine (DPPD), to protect against diquat-induced toxicity in male Fischer-344 rats were examined. Both experimental compounds afforded near complete protection against diquat-induced hepatotoxicity, as measured by clinical chemistry and histopathological indices. When observed, diquat-induced nephrotoxicity was also inhibited. Minimal protection was afforded by the model compound, DPPD. In follow-up studies with U-78517G, no effect on diquat-induced biliary excretion of oxidized glutathione was observed, suggesting that a shift in the thiol:disulfide ratio is not responsible for diquat-induced hepatotoxicity. These data are consistent with those from previous in vitro studies in our laboratory and are in agreement with studies by others which suggest that lipid peroxidation is an important event in diquat-induced hepatotoxicity in vivo. The antioxidant effects were largely route-independent as either oral pre-treatment alone (200 mg/kg, 24 h before diquat), intravenous pre-treatment alone (6 mg/kg, 5 min before diquat) or the combination of both treatments produced a similar degree of protection. While pre-treatment with antioxidants was quite effective, no significant U-78517G-dependent inhibition of toxicity was observed when administration was delayed by as little as 10 min post diquat. These latter data suggest that initiation of diquat-induced hepatotoxicity is rapid and that these compounds would therefore be unlikely to have clinical utility in the treatment of diquat intoxication.

Timoprazole Is a Unique Cytoprotective Agent in the Rat

Timoprazole, a substituted benzimidazole, is an antisecretory agent that inhibits gastric acid secretion by interference with (H+-K+)-ATPase. In the studies reported herein, timoprazole given orally was found to be cytoprotective for the stomach when given 30 min prior to a challenge to boiling water, ethanol, or 0.6 N HCl. Timoprazole also prevented necrosis of the mucosa and acute ulcerations induced by alcohol in the rat fundus, as evaluated by histopathology. The ED50 for cytoprotection was between 1 and 3 mg/kg of timoprazole depending on the challenge, whereas the antisecretory ED50 was approximately 12 mg/kg. Timoprazole was an active antisecretory agent when given subcutaneously (ED50 10 mg/kg), but was not cytoprotective when given by this route. Indomethacin pretreatment (5 mg/kg orally) blocked the cytoprotective activity of oral timoprazole at doses of 1 or 3 mg/kg given 30 min later. However, at higher doses of timoprazole (5 mg/kg), indomethacin did not inhibit the cytoprotective activity. The ability of high doses of timoprazole to overcome the indomethacin blocks is different than the cytoprotective activity of mild irritants, which is always blocked by indomethacin. However, when tested in vitro, timoprazole exhibited only mild inhibitory activity on both prostaglandin cyclooxygenase and 15-hydroxyl-dehydrogenase and only at high doses, suggestive of nonspecific activity.