Richard J. Calvert

Hepatic toxicity of unmodified and time-release preparations of niacin

Niacin (nicotinic acid) is used frequently in the treatment of hypercholesteremia. It is available in both unmodified and time-release preparations. The latter were developed in attempts to minimize the skin-flushing reaction that affects virtually all users and may limit acceptance. Adverse effects on the liver from both unmodified and time-release preparations have been recognized for many years. We reviewed the literature on the hepatic toxicity of both types of niacin preparations. Adverse reactions in six patients resulted from the exclusive use of unmodified niacin and in two patients from the exclusive use of time-release preparations. In 10 additional patients, adverse reactions developed after an abrupt change from unmodified to time-release preparations. Many of these patients were ingesting time-release niacin at doses well above the usual therapeutic doses currently recommended. Signs of liver toxicity developed in less than 7 days in four of these 10 patients. In doses that achieve equivalent reductions in serum lipids, hepatic toxicity occurred more frequently with time-release preparations than with unmodified preparations. An awareness of toxicity associated with ingestion of high doses of time-release niacin preparations is important because of their widespread availability and the potential for self-prescribed, unmonitored use.

Polymerase chain reaction–single‐strand conformation polymorphism analysis for the VHL gene in chemically induced kidney tumors of rats using intron‐derived primers

Von Hippel‐Lindau (VHL) gene mutations occur throughout three exons including the exon‐intron boundaries in human VHL disease–associated and sporadic renal cell carcinomas. To explore the possible role of the VHL gene in chemically induced rat kidney tumors originating from various cell types, more than 150 bp of Fischer 344 and Noble rat VHL intron sequences flanking the three exons was determined by dideoxy sequencing. Five primer sets were selected for polymerase chain reaction amplification of the coding regions of rat VHL exons 1–3 and the exon‐intron boundaries. Tissues from 10 renal eosinophilic epithelial tumors induced by N‐nitrosoethyl(2‐hydroxyethyl)amine, 10 nephroblastomas induced by N‐nitroso‐N‐ethylurea, and seven renal mesenchymal tumors induced by N‐nitrosomethyl(methoxymethyl)amine were examined for VHL mutations by polymerase chain reaction–single‐strand conformation polymorphism analysis. No mutation was detected in any tumor type, indicating that VHL mutations are not involved in the pathogenesis of rat kidney tumors arising from the distal region of the renal tubules, the metanephric blastema, or stromal tissues of the cortex. Mol. Carcinog. 19:230–235, 1997.

Doxorubicin-treated H9c2 cells: caution with luminescent ATP and Hoechst 33258 assays

To the Editor: High throughput, plate-based cellular assays are becoming increasingly popular due to rapidity of data generation and the opportunity for a higher number of replicates than possible with older methods. Assays for DNA content based on Hoechst 33258 (H33258) fluorescence (Rago et al. 1990) and ATP content using a kit based on a series of coupled reactions producing chemiluminescence in response to ATP levels (such as Cell Titer-Glo, Promega, Madison, WI) are popular biomarkers used in high throughput assays. We found when using doxorubicin as a toxicant in initial experiments that H33258 fluorescence was extremely low, while ATP content based on luminescence was higher than expected. To investigate the DNA results, eight concentrations of DNA ranging from 0.024 to 3.0 μg/0.1 ml dissolved in water were incubated with doxorubicin at 1 or 5 μM for 4 h prior to addition of the H33258. Fluorescence at 458 nM (with excitation at 358 nM) was measured as per the published protocol (Rago et al. 1990). Water or 1 or 5 μM doxorubicin-only blanks were subtracted from the raw data. Fluorescence results are shown in Fig. 1. If a point in the figure lacks SEM bars, they were too small to appear outside of the data point. At all DNA concentrations, fluorescence was significantly lower (p<0.0002, Mann–Whitney test) with added doxorubicin at 1 or 5 μM compared to fluorescence without doxorubicin, except at the lowest DNA concentration in the 5-μM doxorubicin group. To investigate apparent enhanced ATP content with doxorubicin treatment, H9c2 cardiomyoblasts (ATCC) seeded in 96-well plates were treated with 1 or 5 μM doxorubicin for 24 h. Results of ATP determination (Cell Titer-Glo, Pomega) were found to be higher when doxorubicin was left in the well vs. replacement with serum-free media just prior to the ATP assay (Fig. 2). To explain these results, we hypothesized that doxorubicin is much more avid in its DNA intercalation than H33258, resulting in lower fluorescence when doxorubicin is present. The ATP results with doxorubicin left in the cell media were likely due to fluorescence emission from doxorubicin excited by the white light luminescence from the ATP-coupled reaction, resulting in spuriously high luminescence values (personal communication with technical representative at Promega 2012). We suggest that the use of another cellularity indicator,

A beating heart cell model to predict cardiotoxicity: effects of the dietary supplement ingredients higenamine, phenylethylamine, ephedrine and caffeine.

Some dietary supplements may contain cardiac stimulants and potential cardiotoxins. In vitro studies may identify ingredients of concern. A beating human cardiomyocyte cell line was used to evaluate cellular effects following phenylethylamine (PEA), higenamine, ephedrine or caffeine treatment. PEA and higenamine exposure levels simulated published blood levels in humans or animals after intravenous administration. Ephedrine and caffeine levels approximated published blood levels following human oral intake. At low or midrange levels, each chemical was examined plus or minus 50 µM caffeine, simulating human blood levels reported after consumption of caffeine-enriched dietary supplements. To measure beats per minute (BPM), peak width, etc., rhythmic rise and fall in intracellular calcium levels following 30 min of treatment was examined. Higenamine 31.3 ng/ml or 313 ng/ml significantly increased BPM in an escalating manner. PEA increased BPM at 0.8 and 8 µg/ml, while 80 µg/ml PEA reduced BPM and widened peaks. Ephedrine produced a significant BPM dose response from 0.5 to 5.0 µM. Caffeine increased BPM only at a toxic level of 250 µM. Adding caffeine to PEA or higenamine but not ephedrine further increased BPM. These in vitro results suggest that additional testing may be warranted in vivo to further evaluate these effects.

Treatment with lovastatin, cholestyramine or niacin alters K-ras membrane association in mouse lung in a strain-dependent manner: results in females.

Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-ras membrane localization, with possible pro- or anti-carcinogenic effects (K-ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3x) or five (Lov-5x) times per week. After three weeks, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-ras proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-ras specific antibody. CS feeding increased membrane K-ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-ras, and resulted in an increase in total K-ras in A/J and C57BL/6 mice, while again lacking effect in Swiss mice. Niacin had no effect on K-ras protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-ras protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less likely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.

Gastrointestinal Effects of Fructooligosaccharides

Fructooligosaccharides (FOS) consist of a glucose (G) unit joined by an (αl-β2) linkage to 2 or more fructose (Fn) units, where n is the number of fructose units. Fructose units are joined to one another by a β(2–1) linkage. Structures for three common FOS are shown in Fig. 1.

Alterations in membrane-bound and cytoplasmic K-ras protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent.

Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) ras protein membrane localization by altering protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25mg/kg of Lov three times per week (Lov-3X) or five times per week (Lov-5X). After 3 weeks, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-ras proteins in lung were determined by immunoprecipitation followed by western blotting with a K-ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-ras and a 40% decrease in membrane K-ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3-28%) of membrane K-ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58-78% increase in cytoplasmic K-ras without any reduction in the levels of membrane K-ras. Niacin, predicted to have no effect on ras membrane localization, decreased cytoplasmic K-ras in A/J mice, increased both membrane and cytoplasmic K-ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-ras membrane localization, and potentially the risk of ras-dependent cancer.

Cardiotoxicity testing of diglycolic acid using In Vitro and In Vivo models

Abstract Background: Renal and hepatotoxicity of diglycolic acid (DGA) has been described with in vitro cellular models as well as in vivo animal and human systems. The possibility of DGA being toxic