Richard A. Galbraith

Drug metabolism by the human hepatoma cell, Hep G2

The human liver-derived cell line, Hep G2, has aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-de-ethylase activities. Partial purification of cytochrome P-450 from Hep G2 cells provided spectral evidence of this hemeprotein in the purified fraction. These results suggest that Hep G2 cells will be useful for the study of cytochrome P-450 and the regulation of mixed function oxidase activities in liver cells of human origin.

The Inhibition of Islet Superoxide Dismutase by Diabetogenic Drugs

The enzyme superoxide dismutase (SOD) is a scavenger of superoxide radicals and protects the integrity of cell membranes. We previously reported that streptozotocin inhibits the SOD activity of erythrocytes and retinae in vivo and in vitro. We now report that the three major diabetogenic drugs, i.e., alloxan, streptozotocin, and Vacor (in order of increasing potency), interact with erythrocyte Cu-Zn SOD in vitro. Maximum inhibition of erythrocyte SOD of man, dog, rat, and cow is 40% and is achieved within 10 min. At submaximal doses the effect of the three drugs is additive. Inhibition induced by streptozotocin, but not by the other two agents, is reversible by dialysis. Alloxan (80 mg/kg), like streptozotocin, also inhibits erythrocyte SOD activity when injected i.v. into rats. Glucose or 3- o-methylglucose does not prevent SOD inhibition by alloxan or streptozotocin in vitro. Injection of glucose before alloxan prevents the development of diabetes, but does not prevent alloxan-lnduced inhibition of erythrocyte SOD. SOD is present in the islets of Langerhans of rats and dogs, as demonstrated by biochemical assay of isolated islets and by immunofluorescent staining of frozen pancreases. The specific activity of SOD in the islets exceeds that of the exocrine pancreas more than 100-fold. The islet SOD is of the Cu-Zn type, since it is inhibited by KCN, and not by chloroformethanol. The mobility of the islet enzyme on polyacrylamlde disc gel electrophoresis is different from that of erythrocyte SOD. Streptozotocin, alloxan, and Vacor Inhibit the activity of islet SOD in vitro; the rate and magnitude of inhibition is the same as that observed with erythrocyte SOD. The Mn SOD of liver mitochondria is not affected by the diabetogenic drugs. It is suggested that the inhibitory effect of the diabeto- genie drugs on islet cell SOD may contribute to their cytotoxicity, and that changes in the amount or activity of this protective enzyme in β-cells may play a role in determining their vulnerability to noxious agents.

The Effects of Cimetidine on the Oxidative Metabolism of Estradiol

Cimetidine, a histamine H2-receptor antagonist widely used to treat peptic ulceration, is known to cause gynecomastia and sexual dysfunction in some men. Since cimetidine inhibits the cytochrome P-450-dependent biotransformation of numerous drugs, we investigated the possibility that it might also inhibit the cytochrome P-450--dependent metabolism of estradiol. Radiometric analysis of urine and serum samples from nine normal male volunteers showed that the extent of 2-hydroxylation of estradiol was significantly reduced from a mean (+/- SEM) of 31.7 +/- 2.3 percent to 19.7 +/- 2.3 percent (P less than 0.0001) after two weeks of oral treatment with cimetidine (800 mg twice a day); the 16 alpha-hydroxylation of estradiol was unaffected. At the same time, the urinary excretion of 2-hydroxyestrone decreased by approximately 25 percent (P less than 0.0002), and the serum concentration of estradiol increased by approximately 20 percent (P less than 0.04). The mean percentage of estradiol 2-hydroxylation was also rapidly reduced, from 36.8 +/- 4.4 percent to 24.5 +/- 3.4 percent in six men after one week of oral cimetidine at a lower dosage (400 mg twice a day; P less than 0.0006). In a separate study of seven men, ranitidine, a second-generation H2-receptor antagonist, was found to have no effect on the 2-hydroxylation of estradiol. This study demonstrates that the administration of cimetidine to men decreases the 2-hydroxylation of estradiol and results in an increase in the serum estradiol concentration. This mechanism may help to account for the signs and symptoms of estrogen excess reported with the long-term use of cimetidine.

Thrombophlebitis and disturbed hemostasis following administration of intravenous hematin in normal volunteers

PURPOSE Acute porphyria episodes are routinely treated with hematin, but side effects, including disturbances of hemostasis and peripheral thrombophlebitis, are associated with the compounds use. Thrombophlebitis is particularly troublesome in patients who require repeated administration of hematin, and may eventually lead to the placement of central venous lines or implantation of indwelling venous access ports. We undertook this study to determine whether only patients with porphyria experienced peripheral thrombophlebitis and disturbed hemostasis following administration of hematin, or if this was a general phenomenon that could also be observed in normal volunteers. SUBJECTS AND METHODS Hematin was administered intravenously in the doses customarily used in therapy of acute porphyria crises (4 mg/kg body weight) to nine normal male volunteers, who were screened by history, physical examination, routine blood cell counts, urinalysis, biochemical screening profile, and coagulation tests. Hemostasis tests were performed in each subject, and hematin concentrations were determined. RESULTS Within the first 24 hours, the activated partial thromboplastin time was prolonged in all subjects (mean of 25 percent), the prothrombin time was increased in eight subjects (mean of 20 percent), and the thrombin time in five subjects rose (mean of 15 percent), whereas the concentration of circulating platelets decreased in three subjects (mean of 20 percent). In four subjects (45 percent), thrombophlebitis developed following hematin infusion. CONCLUSION Although hematin is frequently effective in the treatment of acute porphyria crises, it is often associated with abnormalities in coagulation and these effects also occur in normal volunteers.

Engaging basic scientists in translational research: identifying opportunities, overcoming obstacles

This report is based on the Federation of American Societies for Experimental Biology’s symposium, “Engaging basic Scientists in Translational Research: Identifying Opportunities, Overcoming Obstacles,” held in Chevy Chase, MD, March 24–25, 2011. Meeting participants examined the benefits of engaging basic scientists in translational research, the challenges to their participation in translational research, and the roles that research institutions, funding organizations, professional societies, and scientific publishers can play to address these challenges.

Cimetidine inhibits catechol estrogen metabolism in women

Chronic cimetidine use in men is associated with hyperestrogenic side effects such as gynecomastia, which may be linked to inhibition of estradiol 2-hydroxylation. As this property of the drug might be helpful in hypoestrogenic states such as osteoporosis, we investigated the effect of cimetidine on estradiol metabolism in premenopausal and postmenopausal women. Using an in vivo radiometric assay, we found that the extent of estradiol 2-hydroxylation in premenopausal women (n = 9) was decreased by a 1-month course of cimetidine, 800 mg twice daily (44.0% +/- 3.5% v 31.2% +/- 4.1%, P less than .001). Among premenopausal smokers (n = 3), the response to cimetidine was approximately the same as nonsmokers. Serum estradiol levels (follicular phase) in these women were unaltered by cimetidine after 1 month, while concentrations of sex hormone-binding globulin (SHBG) were decreased by 30% (P = .018). Postmenopausal women (n = 5) initially received a lower dose of cimetidine (600 mg twice daily) for 2 weeks, followed by a higher dose (1200 mg twice daily) for another 2 weeks. The extent of estradiol 2-hydroxylation was significantly reduced by the low dose (44.4% +/- 4.5% v 24.3% +/- 3.0%, P less than .005), with minimal further reduction after the high dose (21.7% +/- 1.6%). After 4 weeks of cimetidine treatment, serum estradiol levels increased significantly from 30.0 +/- 6.4 to 59.8 +/- 13.1 pg/mL (P = .033), while SHBG was unaffected. Cimetidine was found to have little effect on selected biochemical indices of bone and calcium metabolism in both groups of women.(ABSTRACT TRUNCATED AT 250 WORDS)

Tin-Mesoporphyrin Inhibits Heme Oxygenase Activity and Heme-Iron Absorption in the Intestine

Long-term treatment with the heme oxygenase inhibitor tin-mesoporphyrin produces an iron deficiency anemia in rats analogous to that we reported in patients with the Crigler-Najjar type I syndrome receiving prolonged treatment with the inhibitor to ameliorate severe jaundice [Pediatrics 1992; 89: 175-182]. A dose- and time-dependent inhibition of intestinal heme oxygenase is produced by tin-mesoporphyrin which is independent of iron status of the animal. Tin-mesoporphyrin inhibits the intestinal enzyme whether administered orally or parenterally. Enzyme inhibition by either route results in diminished uptake of 59Fe from radiolabelled heme in the gut. Since tin-mesoporphyrin stimulates excretion of unmetabolized heme into bile its ability to inhibit intestinal heme oxygenase and to decrease heme-iron absorption in the gut probably accounts in part for the iron deficiency produced by the agent. The availability of an orally active agent which inhibits heme oxygenase and heme-iron absorption in the intestine may prove useful for experimental and therapeutic studies in diseases of iron metabolism.

Possible role of creatine concentrations in the brain in regulating appetite and weight

Cobaltic protoporphyrin IX (CoPP) is a synthetic heme analog which can elicit profound and prolonged decreases in appetite and body weight in several different animal species. Intracerebroventricular administration of CoPP in rats was found, by differential display and confirmed by Real-Time PCR, to result in an increase in expression of the creatine transporter when compared to vehicle-treated fed or vehicle-treated fasted control animals. In situ hybridization studies showed that creatine transporter mRNA concentrations were increased in several areas of the brain involved in the regulation of food intake, but creatine concentrations were decreased in hypothalamic homogenates in CoPP-treated animals compared to controls. Intracerebroventricular administration of beta-guanidinopropionic acid, a compound known to decrease intracellular creatine concentration by competition for uptake, resulted in decreased food intake and body weight and increased Fos expression in the hypothalamus. Taken together, these findings suggest that creatine concentrations in the brain may play a role in regulating food intake and body weight.

Phenotypic variation in human HepG2 hepatoma cells: Alterations in cell growth, plasma protein synthesis and heme pathway enzymes

1. Growth rates, morphology, plasma protein synthesis and the level of heme pathway enzymes were examined in six sublines of HepG2 cells obtained from various laboratories. 2. Five sublines represented by G2a display the known characteristics of HepG2 cell type, including morphology, plasma protein synthesis and an increase in delta-aminolevulinic acid (ALA) dehydratase activities in response to Me2SO treatment. 3. In contrast, cells of the G2f subline failed to secrete significant quantities of plasma proteins. There was also no increase in ALA dehydratase activity following Me2SO treatment. These findings suggest that G2f cells represent a variant of HepG2 cells with an altered phenotype.

Sifting Robotic from Organic Text: A Natural Language Approach for Detecting Automation on Twitter

Twitter, a popular social media outlet, has evolved into a vast source of linguistic data, rich with opinion, sentiment, and discussion. Due to the increasing popularity of Twitter, its perceived potential for exerting social influence has led to the rise of a diverse community of automatons, commonly referred to as bots. These inorganic and semi-organic Twitter entities can range from the benevolent (e.g., weather-update bots, help-wanted-alert bots) to the malevolent (e.g., spamming messages, advertisements, or radical opinions). Existing detection algorithms typically leverage meta-data (time between tweets, number of followers, etc.) to identify robotic accounts. Here, we present a powerful classification scheme that exclusively uses the natural language text from organic users to provide a criterion for identifying accounts posting automated messages. Since the classifier operates on text alone, it is flexible and may be applied to any textual data beyond the Twitter-sphere.