Mohamed S. Abdel-Rahman

Hepatoprotective activity of thymoquinone in isolated rat hepatocytes

Thymoquinone, the active constituent of Nigella sativa, was tested in isolated rat hepatocytes as a hepatoprotective agent against tert-butyl hydroperoxide (TBHP) toxicity. TBHP (2 mM) was used to produce oxidative injury in isolated rat hepatocytes and caused progressive depletion of intracellular glutathione (GSH), loss of cell viability as evidenced by trypan blue uptake and leakage of cytosolic enzymes, alanine transaminase (ALT) and aspartic transaminase (AST). Preincubation of hepatocytes with 1 mM of either thymoquinone or silybin, which is a known hepatoprotective agent, resulted in the protection of isolated hepatocytes against TBHP induced toxicity evidenced by decreased leakage of ALT and AST, and by decreased trypan blue uptake in comparison to TBHP treated hepatocytes. Both thymoquinone and silybin prevented TBHP induced depletion of GSH to the same extent. Although thymoquinone protected the liver enzymes leakage, the degree of protection was less than that caused by silybin.

Safety evaluation of benzophenone-3 after dermal administration in rats

Benzophenone-3 (BZ-3) is a category 1 (over-the-counter) product approved by the US Food and Drug Administration (FDA) for use as a sunscreen agent in medicine, cosmetics, industry, and agriculture. This is due to its ability to absorb and dissipate ultraviolet light in a harmless manner, thus protecting human skin and products from UV irradiation. This study investigated the safety of BZ-3 after repeated administration. BZ-3 in ointment base was applied at a dose of 100 mg/kg body wt. twice daily, for 4 weeks to the skin of male Sprague-Dawley rats. Body weight, organ to body weight ratios, hematological, and clinical chemistry parameters were not effected. Pathological examination revealed no significant changes between control and treated animals. No gross external abnormalities were observed. Both in vivo and in vitro blood glutathione (GSH) levels were effected by BZ-3 treatment. However, after 60 min of incubation, a reversal of this effect was observed in the treatment group as blood GSH levels approached normal levels. Furthermore, investigation of GSH-reductase and peroxidase with time indicated an increase in GSH-reductase activity at 60 and 90 min with no effect on GSH-peroxidase. Pre-treatment with phenobarbital modulated the metabolic disposition of BZ-3. There was an increase in the formation of the hydroxy metabolites but not the O-dealkylated form. This study suggests that BZ-3 is not toxic to rats when applied dermally at a dose of 100 mg/kg body wt. for 4 weeks.

Cocaine Hepatotoxicity in Mice: Histologic and Enzymatic Studies

The severity of hepatotoxicity in CF-1 mice given 5 daily doses of 5, 10, and 20 mg cocaine/kg body weight and sacrificed 24 hr after the last injection appeared to be dose-dependent. Using light microscopy, the hallmark of cocaine early toxicity was manifested by pallor and ballooning of the hepatocytes in the midzonal and then in the centrilobular areas. Degeneration and necrosis of the liver cells in the same zones were encountered while the hepatocytes in the periportal areas remained intact. When examined under the electron microscope, such pallor and ballooning of the hepatocytes appeared to be due to dilation of the rough endoplasmic reticulum (RER) profiles which of ten revealed a significant loss of their ribosomes. Dilation of the smooth endoplasmic reticulum (SER) was also common and moderate proliferation of peroxisomes was frequently seen. In the degenerating hepatocytes, the 2 forms of endoplasmic reticulum were difficult to recognize and the peroxisomes appeared sparse. Cocaine treatment elevated the level of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase in a dose-dependent manner. Although hepatic cytochrome P450 was slightly increased in the low dose groups, a reduction in the enzyme activity was noticed in the group treated with 20 mg cocaine/kg. However, hepatic reduced glutathione content manifested a significant increase in the group which received 20 mg cocaine/kg.

Evaluation of the use of uncertainty factors in deriving RfDs for some chlorinated compounds.

Risk assessment of exposure to chemicals having a toxic endpoint routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. The purpose of this investigation was to evaluate whether the magnitude of the U.S. Environmental Protection Agency (EPA) 10x uncertainty factors has scientific merit when compared with data from recent human and animal experimental studies. A compilation and comparison of ratios between LOAEL/NOAEL (lowest observed adverse effect level/no observed adverse effect level) and subchronic/chronic values was made for six chlorinated compounds, namely, carbon tetrachloride, methylene chloride, pentachlorophenol, monochlorobenzene, chlorpyrifos, and 1,1-dichloroethane. Data sets demonstrated that 91.3% of the LOAEL/NOAEL ratios were < or = 6 while 87% of the ratios for the same parameter were < or = 5. Furthermore, subchronic/chronic ratios were < or = 3.5. From our investigation we concluded that automatic safety factors of 10x are not scientifically supportable and are overly conservative for the chlorinated compounds studied here.

Oral cocaine produces dose-related hepatotoxicity in male mice

Cocaine remains a widely abused substance. While most addicts take cocaine intranasally, a considerable number abuse cocaine by mouth. It has been assumed that after oral exposure cocaine is hydrolyzed in the stomach rendering it ineffective. This study investigated the effect of orally administered cocaine on liver function and integrity as well as its effect on liver and blood antioxidative enzymes. Male CF-1 mice were orally administered either 0, 5, 10 or 20 mg cocaine/kg body weight and sacrificed 24 h after the last treatment. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as markers of liver injury. Blood and liver glutathione (GSH) levels were determined as well as the activities of glutathione peroxidase (GPx) and catalase (CAT). In addition, the activity of liver glutathione reductase (GRx) was also measured. The results demonstrated that oral cocaine caused hepatotoxicity in a dose dependent manner. Serum ALT and AST were elevated while blood GSH concentration decreased in all cocaine treated animals. In addition, there was a significant dose dependent decrease in the activities of GPx and CAT in blood and liver of cocaine treated animals. However, hepatic GSH content and GRx activity manifested a significant increase, particularly in the group, which received 20 mg/kg cocaine. This study is the first to demonstrate that cocaine-induced hepatotoxicity results following the oral route of administration.

A Comparative Study of the Kinetics and Bioavailability of Pure and Soil-Adsorbed Naphthalene in Dermally Exposed Male Rats

The aim of this study was to utilize pharmacokinetic techniques to assess the bioavailability of sandy or clay soil-adsorbed naphthalene vs chemical alone following dermal treatment of male rats. Animals were exposed to 43 μg total of 14C-naphthalene (pure or adsorbed to one of two soils) introduced into a shallow glass cap covering a 13-cm2 area on the skin of each rat. While both soils delayed the time to reach maximum plasma concentration of radioactivity and significantly increased the half-life of plasma absorption, only sandy soil significantly decreased the peak plasma concentration of radioactivity versus the pure compound. Within 12 h after dermal application, approximately 50% of the naphthalene dose was excreted in the urine of the pure and clay soil-adsorbed groups. However, when naphthalene was adsorbed to sandy soil, the percentages of the initial dose excreted in the urine collected between 0–12 h and 12–24 h were nearly equal (33–39%). Furthermore, sandy soil adsorption shifted the secondary excretion route from expired air to feces and significantly lowered the amount of radioactivity in expired air relative to naphthalene alone. In the presence of sandy soil, a significantly larger amount of radioactivity washed off of the skin application sites. In all groups the predominant urinary metabolites determined by high performance liquid chromatography were 2,7- and 1,2-dihydroxynaphthalenes.

Endotoxin potentiates the hepatotoxicity of cocaine in male mice.

Cocaine produces hepatotoxicity by a mechanism that remains undefined but that has been linked to its oxidative metabolism. Endotoxin (lipopolysaccharide, LPS) is also a well-known cause of hepatic damage, where exposure to non-injurious doses of LPS increases the toxicity of certain hepatotoxins. This study was conducted to investigate the possible potentiation of cocaine-mediated hepatotoxicity (CMH) by LPS. Male CF-1 mice were administered oral cocaine hydrochloride for 5 consecutive days at a dose of 20 mg/kg with and without 12 2 10 6 EU LPS/kg given intraperitoneally 4 h after the last cocaine injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as markers of liver injury. Blood and liver glutathione (GSH) levels were determined, as well as the activities of glutathione peroxidase (GPx) and catalase (CAT). In addition, the activity of liver glutathione reductase (GRx) was measured. The results demonstrate that endotoxin potentiated the hepatotoxicity of cocaine. Serum ALT and AST were significantly elevated with the combined cocaine and LPS treatment versus all other treatments. While cocaine alone resulted in centrilobular necrosis, the cocaine and LPS combination produced submassive necrosis. The increased hepatic GSH content and GRx activity observed with cocaine alone were not observed with the combination treatment, rendering the liver more susceptible to oxidative stress. Moreover, there was a significant decrease in the activities of hepatic GPx and CAT, particularly with the combination treatment. In conclusion, this study demonstrates that LPS potentiates the hepatotoxicity of cocaine as revealed by an array of biochemical and morphological markers.

The mechanism of methyl mercury toxicity in isolated rat hepatocytes.

Mercury is the major component of dental amalgam restorative material, which typically has 50% pure elemental mercury. It is also used in some skin creams, and in the manufacturing of plastic, drugs and fungicides. The present study was designed to investigate the toxicity of methyl mercury (MeHg+) on isolated rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated with different concentrations of MeHg+ (0.1-100 ppm) for 2 h. Through the incubation period the viability was determined by Trypan blue exclusion. Reduced glutathione (GSH) content and its enzymes, glutathione peroxidase (GSH-PX) and glutathione reductase (GSH-RX) were measured. Leakage of enzymes such as aspartate transaminase (AST), and alanine transaminase (ALT) were determined. The cell viability was reduced significantly after 1 h incubation when 0.1 and 1 ppm MeHg+ were applied. The decrease in the cell viability was dose- and time-dependent. A depletion of GSH content was observed with 100 ppm MeHg+ after 30 min of incubation. A significant decrease in GSH-RX was observed with 100 ppm during 15 and 30 min of incubation, while 10 ppm of MeHg+ significantly increased ALT leakage after 60 min. However, there was a significant increase in AST leakage with 100 ppm only. The present investigation indicates that the toxic effect of MeHg+ is most likely cytosolic enzyme related.

Effects of soil on the dermal bioavailability of m-Xylene in male rats

Bioavailability of a chemical absorbed through the skin from contaminated soil may differ from that seen following exposure to the pure chemical. The objective of this research was to qualitatively and quantitatively assess the absorption, distribution, excretion, and metabolism of soil-adsorbed m-xylene versus pure m-xylene so that the potential for public health risk following dermal exposure could be evaluated. In this study a shallow glass cap covering a 13-cm2 area was fixed to the shaved skin of each male rat (6-10 rats per group) followed by the addition of 225 microliters of m-xylene containing 20 muCi of m-[14C]xylene alone or with one of two soils. Maximum plasma levels of radioactivity were highest for pure m-xylene while the values for the sandy and clay soil groups were approximately equal. Although clay soil statistically decreased the rate of absorption, the half-lives of elimination and the area under the plasma concentration time curve were not changed by either soil. The major route of excretion in the pure and sandy groups was via expired air followed by urine. However, in the presence of clay soil, the percentage of the initial dose in expired air was similar to that in urine. Forty-eight hours after treatment, skin application sites in both soil treatment groups contained amounts of radioactivity significantly higher than those of m-xylene treatment alone. In the presence of clay soil a statistical increase in m-xylene-derived radioactivity was also observed in fat beneath the treated skin area. Metabolite analysis by HPLC indicated that methylhippuric acid was the main urinary metabolite followed by xylenol and the parent compound in all groups.

Inhibition of cocaine oxidative metabolism attenuates endotoxin potentiation of cocaine mediated hepatotoxicity

The oxidative metabolism of cocaine by the microsomal monooxygenase enzymes has been postulated to be essential for cocaine mediated hepatotoxicity (CMH). Endotoxin (lipopolysaccharide, LPS), a well-known cause of hepatic damage, previously has been demonstrated to dramatically increase CMH. The mechanism of this interaction has not been clearly elucidated, but cocaine oxidative metabolism appears to sensitize hepatocytes so that subsequent exposure to small amounts of LPS can further augment CMH. This study was conducted to investigate if dimethylaminoethyl-2,2-diphenylvalerate (SKF-525A) pretreatment inhibits LPS potentiation of CMH. For 5 consecutive days, male CF-1 mice were administered daily SKF-525A (50 mg/kg) or sterile saline followed an hour later by cocaine (20 mg/kg) or sterile saline. Four hours following the last cocaine or saline treatment, the mice were administered sterile saline 12x10(6) EU LPS/kg, i.p. The mice were sacrificed 18 h later by decapitation. Pretreatment with SKF-525A reversed the hepatic injury caused by cocaine alone or cocaine and LPS treatments, as indicated by both histologic evaluation and serum alanine transaminase (ALT) and aspartate transaminase (AST) activities. In particular, SKF-525A completely reversed the effects of cocaine alone on liver and blood reduced gluthathione (GSH), glutathione peroxidase (GPx) and catalase (CAT) and hepatic glutathione reductase (GRx) activities. However, SKF-525A was ineffective against the effect of LPS alone on liver and blood GPx and CAT or on hepatic GSH and GRx, suggesting that these effects were not mediated by cytochrome P450 oxidative metabolism. The pattern of biochemical changes persisting with SKF-525A pretreatment in the LPS and cocaine group resembled those of the LPS alone group. The results suggest that cytochrome P450 oxidative metabolism of cocaine is largely responsible for CMH with potentiation by LPS achieved through a different mechanism involving oxidative stress.