Protective and Ameliorative Effects of Hydroethanolic Extract of Piper nigrum (L.) Stem against Antiretroviral Therapy-Induced Hepatotoxicity and Dyslipidemia in Wistar Rats.

Antiretroviral therapy (ART) has revolutionized the lives of people living with HIV/AIDS by overall improving their quality of life and increasing life expectancy. However, ART-associated hepatotoxicity and metabolic disorders in HIV/AIDS patients are growing concerns to clinicians, especially due to the long-term use of the drugs. This study reported on the phytochemical and pharmacological profile of hydroethanolic extracts of Piper nigrum stem (PNS) and evaluated its protective effect against tenofovir/lamivudine/efavirenz (TLE)-induced hepatotoxicity and dyslipidemia in Wistar rats. Cytotoxic, antioxidant, and anti-inflammatory assays were performed on PNS. Thirty-six rats divided into 6 groups of 6 animals/group were administered: distilled water, 17 mg/kg TLE, 17 mg/kg TLE and 100 mg/kg silymarin, 17 mg/kg TLE, and Piper extract (200 mg/kg, 400 mg/kg, or 800 mg/kg) orally for 28 days. The body weight of animals was recorded every 7 days. On Day 29, the rats were sacrificed, and blood samples were collected for hematological and biochemical tests. Portions of the liver and kidneys were collected for histological evaluation, while liver homogenates were prepared from the rest to measure antioxidant enzymes. PNS possessed in vitro cytotoxic, antioxidant, and anti-inflammatory activities. A significant decrease (p < 0.05) in the body weight of rats treated with PNS was observed. A significant high platelet count (p < 0.05) was observed in the PNS800 mg/kg group. A considerable decrease in alkaline phosphatase and triglycerides was observed in the silymarin and PNS group compared to the TLE-only group. The findings also show a significant increase in catalase and glutathione in the TLE-only group compared to the normal group, while SOD decreased. Histological observations revealed normal hepatic and renal tissues in the silymarin, and PNS-treated groups compared to the normal control, while leucocyte infiltration was observed in the TLE-only group. These results suggest that PNS extract possessed antioxidant activity that alleviated TLE-induced toxicity. Further studies are necessary to understand the pharmacokinetic interactions between ART and PNS.

Pharmacological and toxicological effects of chronic porcine growth hormone administration in dogs.

The purpose of this study was to evaluate the pharmacological and toxicological effects of exogenous GH administration in normal adult dogs. Because porcine GH (pGH) is structurally identical to canine GH, pGH was selected for a 14-wk study in dogs. Thirty-two dogs (< 2 yr) were randomized to 4 groups (4 dogs/sex/group); 1 group was treated with the vehicle and 3 groups received pGH at 0.025, 0.1, or 1.0 IU/kg/day subcutaneously. Daily clinical signs and weekly body weights were recorded. Hematology, serum biochemistry, urinalyses, electrocardiograms, and ophthalmoscopic examinations were done. Serum GH, insulin-like growth factor-1 (IGF-1), insulin, thyroxine (T4), triiodothyronine (T3), and cortisol levels were determined. Necropsies were performed, organs weighed, and tissues were fixed and processed for light microscopic examination. Porcine GH caused increased body weight gain (p < or = 0.05) through the mid dose; the mean weight gains at study termination in mid- and high-dose groups were 2.8 kg and 4.7 kg, respectively, compared to 0.4 kg and 0.8 kg in control and low-dose groups, respectively. Dose-related increased weights of liver, kidney, thyroid, pituitary gland, skeletal muscle, and adrenal gland were noted. In pGH-treated dogs, increased skin thickness seen grossly correlated histologically with increased dermal collagen. There was no gross or histomorphological evidence of edema. There were dose-related increased serum IGF-1 levels (approximately 2-10-fold; p < or = 0.05) that correlated with the elevated serum GH levels in pGH-treated dogs. Also, increased serum insulin levels (p < or = 0.05) through the mid dose were seen throughout the study. In high-dose dogs, the insulin levels remained elevated over 24 hr postdose. The serum glucose levels in fasted dogs remained within the control range and there was no chronic hyperglycemia based on glycosylated hemoglobin levels. Renal glomerular changes, significant polyuria with decreased urine specific gravity, and increased serum insulin levels suggested that the dogs had early insulin-resistant diabetes. There was minimal or no biologically significant effect of pGH on serum T3, T4, and cortisol levels in dogs. Other serum biochemical changes in pGH-treated dogs included decreased urea nitrogen and creatinine, and increased potassium, cholesterol, and triglycerides. Significant increases in serum calcium and phosphorous levels and alkaline phosphatase activity (bone isozyme) correlated with the histological changes in bone. In pGH-treated dogs, there was a dose-related normochromic, normocytic, nonregenerative anemia. The changes described above, except for the anemia, are related to either anabolic or catabolic effects of high doses of GH. Based on this study, it is concluded that the dog is a good model in which to evaluate the safety of GH secretagogues as well as compounds with GH-like activity.

Hepatotoxicity of an HIV protease inhibitor in dogs and rats.

Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as alpha-naphtylisothiocyanate and ethinyl estradiol.

Lipidosis induced in the dog gallbladder by a direct 5-lipoxygenase inhibitor.

The direct 5-lipoxygenase inhibitor L-739,010 was administered at a dose of 60 mg/kg/day per os to beagle dogs for 15 days. Histopathological examination of gallbladders from treated dogs showed epithelial vacuolation and submucosal infiltration by foamy macrophages that were positive for lipids in Sudan Black-and/or Oil Red O-stained sections. Scanning electron microscopic examination of gallbladder mucosa showed thickening of epithelial folds and multifocal epithelial membrane disruptions. Transmission electron microscopy confirmed these findings and showed mucosal epithelial cell lipid droplet accumulation and submucosal infiltration of macrophages filled with lipid droplets, myelin figures, heterophagosomes, and cholesterol clefts. These changes resembled those reportedly seen in the human gallbladder with cholesterolosis and/or chronic cholecystitis.

CYP1A1 specificity of Verlukast epoxidation in mice, rats, rhesus monkeys, and humans.

It has previously been shown that Verlukast is converted to Verlukast dihydrodiol in microsomes from beta-naphthoflavone (BNF)-treated, but not uninduced Swiss Webster mice and Sprague-Dawley rats. We have examined the involvement of CYP1A1 in this reaction in more detail. It is concluded that this reaction is catalyzed exclusively by CYP1A1 in rats, mice, and humans based on the following criteria: 1) the epoxidation of Verlukast is negligible in uninduced rats, which express CYP1A2 but not CYP1A1; 2) Verlukast epoxidation is highly inducible by BNF treatment (60- to 200-fold); 3) Verlukast epoxidation in BNF-treated rat microsomes was inhibited by alpha-naphthoflavone (ANF) treatment, indicating that this activity was mediated by the CYP1A subfamily; 4) > 95% of Verlukast epoxidation in BNF-treated rat microsomes was inhibited by antibodies raised against CYP1A1; and 5) Verlukast was epoxidized by human CYP1A1 but not CYP1A2. Thus, Verlukast epoxidation appears to be specific for rat, mouse, and human CYP1A1. Additional studies showed that Verlukast was metabolized to Verlukast dihydrodiol in microsomes from uninduced rhesus monkeys. This reaction was inhibited by nanomolar concentrations of ANF in rhesus monkey microsomes implicating the involvement of the CYP1A subfamily. In addition, the 8-hydroxylation of R-warfarin, a pathway that is selective for rodent and human CYP1A1 activity, was also catalyzed at significant rates by rhesus monkey microsomes. These findings indicate that, unlike rats, mice, and humans, which have very low constitutive levels of hepatic CYP1A1 activity, the uninduced rhesus monkey is able to catalyze reactions specific to CYP1A1 in rodents and humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Enantioselective induction of peroxisomal proliferation in CD-1 mice by leukotriene antagonists.

The effects of a racemic leukotriene antagonist (MK-0571) and its component enantiomers (L-668,018 and L-668,019) on hepatic peroxisome proliferation were examined in mice, rats, and rhesus monkeys. Administration of racemic MK-0571 to mice resulted in increased liver weights, increased peroxisomal volume density, and a pleiotropic induction of characteristic peroxisomal and nonperoxisomal enzyme activities associated with peroxisomal proliferation. When the individual enantiomers of MK-0571 were administered to mice, a pronounced enantioselective induction of peroxisome proliferation was observed. Toxicokinetic studies showed that the levels of each enantiomer in the liver or plasma after separate administration were similar. Thus, the enantioselectivity in the induction of peroxisome proliferation could not be explained on the basis of pharmacokinetic differences between the enantiomers. The hepatic peroxisomal response of the rat to MK-0571 was greatly attenuated compared to the mouse. As has been seen with other peroxisome-proliferating agents, MK-0571 had no effect on either peroxisomal volume density or peroxisomal enzyme activity in monkeys. Due to the high degree of enantiomeric discrimination toward the induction of peroxisomal proliferation by these enantiomers, compounds of this type may prove useful as probes to examine the mechanisms by which peroxisomal proliferating agents induce their effects.

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.

Thiobenzamide-induced hepatotoxicity: effects of substituents and route of administration on the nature and extent of liver injury.

Differences in the nature and extent of hepatic injury were examined after administration of para-substituted thiobenzamides to rats. In accordance with previous studies, the extent of hepatotoxicity varied with the electron-donating ability of the substituent. There was also a good correlation between the extent of hepatic necrosis and the amount of substituted thiobenzamide sulfoxide found in the plasma after intraperitoneal dosing. The nature of the hepatic lesion, characterized as a combination of hepatic necrosis, ballooning degeneration, and biliary dysfunction, varied qualitatively with each thiobenzamide analog. When the hepatotoxicity of thiobenzamide was compared after either intraperitoneal or oral dosing, differences in the extent of hepatic necrosis, ballooning degeneration, transaminase elevation, and biliary dysfunction were observed. Intraperitoneal dosing with thiobenzamide gave less severe necrosis and more pronounced elevations in bile acids, while oral dosing led to more severe necrosis along with impaired biliary function. The route of administration was shown to dramatically affect the pharmacokinetics of thiobenzamide and thiobenzamide sulfoxide. Intraperitoneal administration of thiobenzamide gave high plasma and liver levels of both thiobenzamide and thiobenzamide sulfoxide, whereas oral administration gave slightly lower levels of the sulfoxide but much lower levels of thiobenzamide. The reason for greater hepatic necrosis after oral administration may be due to a greater ability to further metabolize the sulfoxide to a reactive metabolite in the absence of high levels of thiobenzamide.

Studies on the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on the rodent forestomach.

A novel class of hypocholesterolaemic agents, HMG CoA reductase inhibitors, was shown to cause mucosal thickening in the rodent (mouse and rat) forestomach after subacute/subchronic oral administration. These changes were characterized histologically by acanthosis and hyperkeratosis of the squamous epithelium with submucosal oedema and occasionally cellular infiltration. This drug-induced hyperplastic response was both dose and time dependent, did not occur after subcutaneous administration, and was confined entirely to the rodent forestomach (not observed in any other area of the gastro-intestinal tract). The forestomach hyperplastic response correlated with the pharmacological potency of HMG CoA reductase inhibitors of similar structure (observed to varying degrees with all HMG CoA reductase inhibitors examined to date).

Natural history of the obstructed rabbit appendix: observations with radiography, sonography, and CT.

Chronic (1-3 months' duration) appendiceal obstruction was induced in 11 rabbits to assess the pathologic consequences and to study the associated radiologic, sonographic, and CT findings. Three pathologic/radiologic groups resulted with approximately equal frequency. In group A, the abscess was characterized by inflammatory cells in the lumen and wall of the appendix without mucin production. Calcification was shown radiographically, and sonography showed an anechoic or complex pattern. Group B, the "mixed response," was characterized by an intact hyperplastic mucosa, mucin secretion, and inflammatory debris in the lumen. Occasional calcification was present radiographically, and sonography showed a complex or hypoechoic pattern. In group C, true mucoceles had an intact hyperplastic mucosa, a mucin-filled lumen, and minimal inflammation. These were anechoic on sonography except for mobile foci of inflammatory debris. Chronic obstruction of the appendix results in a spectrum of pathologic responses with varying degrees of either inflammation and mucosal destruction or mucosal hyperplasia and mucin secretion. An abscess results when infection overwhelms the host's inflammatory responses. If the bacteria are destroyed by these defenses, a mucocele forms. An intermediate situation occurs when there is a mixed response with chronic inflammatory changes and an intact mucosa. This finding supports the existence of chronic appendicitis in humans.

Prevention of experimental allergic encephalomyelitis by ganglioside GM4. A follow-up study.

The myelin-specific ganglioside GM4, when incubated with myelin basic protein prior to inoculation, prevents experimental allergic encephalomyelitis in the guinea pig. The monosialoganglioside GM1, the predominant ganglioside of mammalian myelin, also prevents experimental allergic encephalomyelitis, whereas a mixture of neural gangliosides rich in polysialogangliosides causes enhanced disease. Guinea pigs inoculated with both myelin basic protein and GM4 develop serum IgG antibodies against basic protein, but not against GM4.

Evaluation of the Angelchik antireflux prosthesis using a model for esophageal reflux in rhesus monkeys.

Minimal data are available about the Angelchik antireflux prosthesis although it has been inserted in more than 14,000 patients. The present animal study was designed to evaluate the effectiveness and mechanism of action of this prosthesis. A reproducible model of esophageal reflux in primates was created using a double myotomy. Lower esophageal sphincter (LES) pressure and reflux score were improved significantly in animals by insertion of an Angelchik antireflux prosthesis, a modified antireflux prosthesis, or a Nissen fundoplication. Manometrically determined LES length was increased after insertion of an Angelchik antireflux prosthesis but not by a Nissen fundoplication or sham operation. Complications after insertion of the modified prosthesis included intraluminal erosion, fibrous stricture, and slippage of the device over the stomach.

Rib fracture healing in experimental flail chest.

Sixteen dogs were placed under general anesthesia and flail segments of the left chest were created by transecting ribs 7,8,9, and 10 anteriorly and posteriorly. Fractures were 10 cm apart so that a 10-cm flail segment encompassing four ribs was created. In Group I, the control (N = 5), the chest wall muscles were closed without any stabilization of the fractures. Group II (N = 5) had stabilization of both anterior and posterior fracture sites by polypropylene sutures. Group III (N = 6) had stabilization of the fractures in ribs 7 and 8 with 2.5-cm bone grafts taken from the left fourth rib. Ribs 9 and 10 were stabilized by polypropylene sutures. The study established a canine model for flail chest. It also showed that strut stabilization of rib fractures with bone grafts promotes better healing than suture stabilization. It suggests that using bone grafts taken from another rib to stabilize flail segments may be unsatisfactory since the rib used as a donor showed no signs of regeneration at 30 days.

Evaluation of the zetacrit and zeta sedimentation ratio in dogs.

The Wintrobe erythrocyte sedimentation rate (ESR) has been used in animals and persons as a screening test for inflammatory disease and as a monitor for disease progression in individuals. Now, the zetacrit (ZC) and zeta sedimentation ratio (ZSR) are suggested as suitable rapid laboratory tests to replace the ESR in dogs. In 65 dogs, a strong inverse correlation between ESR and ZC was observed (r = -0.961, P less than 0.05). Significant correlations of similar magnitude between ZC and hematocrit (r = 0.587, P less than 0.05) and ESR and Hct (r = 0.569, P less than 0.05) were observed. In addition, influences of various protein fractions, such as fibrinogen, globulin, and albumin, on the ESR and ZC were evaluated and found to be of similar magnitude. These data indicate that ESR and ZC are equivalent methods for determining erythrocyte sedimentation rates in dogs. A strong correlation (r = 0.900, P less than 0.05) between Wintrobe ESR, adjusted for the effect of Hct (ESRHct), and ZSR was observed. The influence of plasma proteins on ESRHct and ZSR was of similar magnitude. However, a weak yet significant correlation between ESRHct and the microhematocrit remained (r = 0.291, P less than 0.05). Data indicate that the latter more effectively factors out the influence of Hct on sedimentation.

Pathogenicity of retroviruses containing either the normal human c-Ha-ras1 gene or its mutated form derived from the bladder carcinoma EJ/T24 cell line.

Cellular ras oncogenes transduced by retroviruses carry mutations in amino acids 12, 59 and 122. Similar mutations have been observed in ras oncogenes activated during induction of neoplasia in both humans and experimental animals. The unmutated normal rat or human c-Ha-ras-1 genes have the ability to transform NIH 3T3 cells in culture when activated by a RNA synthesis promoter. These findings raise the question of whether the mutations are necessary for the ras oncogenes to induce the neoplastic phenotype in vivo. To address this question, we inserted the normal human c-Ha-ras-1 or its mutated counterpart EJ/T24 bladder carcinoma oncogene independently into a retrovirus vector derived from the M1 strain of Moloney murine sarcoma virus (MoMuSV). Both recombinant clones induced foci of transformed cells in an NIH 3T3 cell transfection assay. Infectious virus particles were rescued from cloned transformants carrying a single copy of the integrated provirus using the nonpathogenic amphotrophic wild mouse leukemia virus (WMLV) as helper. The pseudotypes rescued from the EJ/T24-containing transformants had higher titers than the normal c-Ha-ras-1 pseudotypes as determined by a focus assay and gave rise to larger and earlier detected foci upon infection of NIH 3T3 cells. The two pseudotypes were tested for in vivo pathogenicity by inoculation into newborn NFS mice and were compared to the pseudotype WMLV/Harvey murine sarcoma virus (HaMuSV) (positive control) and WMLV (negative control). While the WMLV/EJ/T24 and the WMLV/HaMuSV pseudotypes induced erythroleukemias and sarcomas with a latency period of 6-9 weeks, the WMLV/c-Ha-ras-1 pseudotype induced only mild splenomegaly. As expected the WMLV negative control induced no pathology. Tumor-bearing animals that were not euthanized at 6-9 weeks died within 2-3 months following virus inoculation.

Megadose corticosteroid therapy following experimental traumatic spinal injury.

Corticosteroids are frequently used in the treatment of spinal trauma, although neither experimental nor clinical evidence to support their use is persuasive. Recently there have been claims that extremely high doses ("megadoses") of corticosteroids (equivalent to 15 to 30 mg/kg of methylprednisolone) improve neurological recovery compared to results with traditional steroid doses. The authors have compared the effect of megadose dexamethasone and methylprednisolone therapy to that of saline treatment following traumatic cervical spinal injury in the cat. During 6 weeks postinjury, neurological recovery did not differ significantly in corticosteroid-treated and saline-treated animals. Moreover, histopathological changes in the spinal cord were similar in methylprednisolone- and saline-treated cats. Corticosteroid-treated animals had a higher mortality rate than did control animals, with the predominant cause of death being neurogenic pulmonary edema. It is concluded that megadose corticosteroid treatment does not improve neurological recovery in this experimental model of spinal injury, and is associated with increased mortality.

Prevention of experimental allergic encephalomyelitis by ganglioside GM4.

Guinea pigs inoculated with a mixture of myelin basic protein and the myelin-specific ganglioside, sialosylgalactosylceramide (GM4), do not develop the signs or neuropathology of experimental allergic encephalomyelitis. The results indicate that GM4 cloaks the basic protein molecule so that it is no longer immunopathogenic in these animals. The interaction of basic protein with GM4, previously shown in vitro, appears to be relevant to the pathogenesis of experimental allergic encephalomyelitis in guinea pigs.