Body fat in children does not adversely influence bone development: a 7-year longitudinal study (EarlyBird 18).

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Both negative and positive associations have been reported between body fat and bone density. Extra mechanical loading from excess fat may lead to greater bone mass. Excess ectopic fat may lead to bone demineralisation through inflammatory pathways. WHAT THIS STUDY ADDS: Longitudinally collected data from narrow-angle beam densitometry gives a novel insight into bone growth through adolescence. There is no evidence of a deleterious effect of body fat on children's growing bones after adjustment for height and age. Body fat, mediated by puberty, is associated with larger bones in boys and bones that are both denser and larger in girls. OBJECTIVE: Bone growth is an important determinant of peak bone mass and fracture risk, but there is limited data on the impact of fat-on-bone development at a time when childhood obesity is reaching epidemic proportions. Accordingly, we explored the effect of body fat (BF) on bone growth over time in the context of age, pubertal tempo and gender. METHOD: A cohort of 307 children was measured biannually from 9-16 years for height and weight, and every 12 months for percent BF, bone area (BA), bone mineral content and areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry. Pubertal tempo was determined quantitatively by age at peak height velocity. RESULTS: Percent BF increased and then fell in the boys, but increased throughout in the girls. aBMD and BA increased in both genders (P < 0.001). Greater BF was associated with higher aBMD and BA in girls (P < 0.001), but only BA in boys (P < 0.001). The extra aBMD associated with increased BF was greater in older girls. The rise in aBMD and BA was associated with earlier puberty in both genders (P < 0.001). The impact of BF on aBMD was greater in later puberty in girls (0.0025 g cm(-2) per 10% BF at 10 years versus 0.016 g cm(-2) per 10% BF at 14 years, P < 0.001). CONCLUSION: Greater BF is associated with larger bones, but also denser bones in girls. The effects of fat and puberty are complex and gender specific, but BF of contemporary UK children does not appear to be deleterious to bone quality.

The novel, orally active, delta opioid RWJ-394674 is biotransformed to the potent mu opioid RWJ-413216.

Although the mu opioid receptor is the primary target of marketed opioid analgesics, several studies suggest the advantageous effect of combinations of mu and delta opioids. The novel compound RWJ-394674 [N,N-diethyl-4-[(8-phenethyl-8-azabicyclo]3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide]; bound with high affinity to the delta opioid receptor (0.2 nM) and with weaker affinity to the mu opioid receptor (72 nM). 5'-O-(3-[(35)S]-thio)triphosphate binding assay demonstrated its delta agonist function. Surprisingly given this pharmacologic profile, RWJ-394674 exhibited potent oral antinociception (ED(50) = 10.5 micromol/kg or 5 mg/kg) in the mouse hot-plate (48 degrees C) test and produced a moderate Straub tail. Antagonist studies in the more stringent 55 degrees C hot-plate test demonstrated the antinociception produced by RWJ-394674 to be sensitive to the nonselective opioid antagonist naloxone as well as to the delta- and mu-selective antagonists, naltrindole and beta-funaltrexamine, respectively. In vitro studies demonstrated that RWJ-394674 was metabolized by hepatic microsomes to its N-desethyl analog, RWJ-413216 [N-ethyl-4-[(8-phenethyl-8-azabicyclo[3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide], which, in contrast to RWJ-394674, had a high affinity for the mu rather than the delta opioid receptor and was an agonist at both. Pharmacokinetic studies in the rat revealed that oral administration of RWJ-394674 rapidly gave rise to detectable plasma levels of RWJ-413216, which reached levels equivalent to those of RWJ-394674 by 1 h. RWJ-413216 itself demonstrated a potent oral antinociceptive effect. Thus, RWJ-394674 is a delta opioid receptor agonist that appears to augment its antinociceptive effect through biotransformation to a novel mu opioid receptor-selective agonist.

Topiramate and phenytoin pharmacokinetics during repetitive monotherapy and combination therapy to epileptic patients.

PURPOSE: To evaluate the potential pharmacokinetic interactions between topiramate (TPM) and phenytoin (PHT) in patients with epilepsy by studying their pharmacokinetics (PK) after monotherapy and concomitant TPM/PHT treatment. METHODS: Twelve patients with epilepsy stabilized on PHT monotherapy were enrolled in this study, with 10 and seven patients completing the phases with 400 and 800 mg TPM daily doses, respectively. TPM was added at escalating doses, and after stabilization at the highest tolerated TPM dose, PHT doses were tapered. Serial blood and urine samples were collected for PK analysis during the monotherapy phase or the lowest PHT dose after taper and the concomitant TPM/PHT phase. Potential metabolic interaction between PHT and TPM also was studied in vitro in human liver microsomal preparations. RESULTS: In nine of the 12 patients, PHT plasma concentrations remained stable, with a mean (+/-SD) area under the curve (AUC) ratio (combination therapy/monotherapy) of 1.13 +/- 0.17 (range, 0.89-1.23). Three patients had AUC ratios of 1.25, 1.39, and 1.55, respectively, and with the addition of TPM (800, 400, and 400 mg daily, respectively), their peak PHT plasma concentrations increased from 15 to 21 mg/L, 28 to 36 mg/L, and 27 to 41 mg/L, respectively. Human liver microsomal studies with S-mephenytoin showed that TPM partially inhibited CYP2C19 at very high concentrations of 300 microM (11% inhibition) and 900 microM (29% inhibition). Such high plasma concentrations would correspond to doses in humans that are 5 to 15 times higher than the recommended dose (200-400 mg). TPM clearance was approximately twofold higher during concomitant TPM/PHT therapy CONCLUSIONS: This study provides evidence that the addition of TPM to PHT generally does not cause clinically significant PK interaction. PHT induces the metabolism of TPM, causing increased TPM clearance, which may require TPM dose adjustments when PHT therapy is added or is discontinued. TPM may affect PHT concentrations in a few patients because of inhibition by TPM of the CYP2C19-mediated minor metabolic pathway of PHT.

Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.

INTRODUCTION: The aim of this investigation was to evaluate methods for increasing Caco-2 cell throughput for assessing drug intestinal absorption. The use of 6-, 12-, and 24-well membranes and the effect of membrane size on permeability and the integrity of the Caco-2 cell monolayer were assessed. In an effort to optimize the assessment of drug permeability, increased throughput was investigated by testing compounds singly or as mixtures of analytes. METHOD: The transepithelial electrical resistance (TEER) of cell monolayers was measured on 0.33, 1.0, and 4.7 cm2 polycarbonate membranes using EVOM, over a 25-day period. Absorptive transport was determined on all compounds tested using LC-MS/MS assays, or liquid scintillation spectrometry. RESULTS: The effect of multiple compounds in one well compared to single compounds was assessed with atenolol, nadolol, metoprolol, and propranolol for mixtures of four compounds and with RWJ-53308, atenolol, terbutaline, propranolol, naproxen, piroxicam, topiramate, and furosemide for mixtures of eight compounds. The apparent permeability (Papp) values correlated well between single analytes and mixtures of four and eight analytes in each well. Drug permeability decreased slightly with an increase in well size. The TEER value increased with the number of days in culture for each of the 6-, 12-, and 24-well sizes. DISCUSSION: It was demonstrated that the 24-well format system is ideal for high-throughput assessment. Furthermore, the approach of mixing four or eight analytes in each well to further increase throughput was also demonstrated to be valid.

An overview of the preclinical aspects of topiramate: pharmacology, pharmacokinetics, and mechanism of action.

In this overview, we discuss the discovery and development of topiramate (TPM) as an anticonvulsant, including notable aspects of its chemical, biologic, and pharmacokinetic properties. In particular, we highlight its anticonvulsant profile in traditional seizure tests and animal models of epilepsy and the results of recent electrophysiological and biochemical studies using cultured neurons that have revealed a unique combination of pharmacologic properties of TPM. Finally, we present a hypothesis for the mechanistic basis of the anticonvulsant activity of TPM, which proposes that TPM binds to certain membrane ion channel proteins at phosphorylation sites and thereby allosterically modulates channel conductance and secondarily inhibits protein phosphorylation.

Pharmacokinetics and bioavailability of topiramate in the beagle dog.

Male and female beagle dogs showed rapid absorption following oral administration of single oral gavage (40 mg/kg) and single or multiple (15 days) oral capsule (10, 40, and 150 mg/kg) doses of the novel anticonvulsant drug, topiramate, with the peak plasma concentration (Cmax) occurring between 0.6 and 3.8 hr. The absolute bioavailability of an oral dose of topiramate was estimated to be in the range of 27-59%, depending on the formulation. The mean topiramate Cmax values increased in a dose-proportional manner for both single (9.2-137.7 micrograms/ml) and multiple (10.3-145.2 micrograms/ml) oral capsule administrations, whereas the corresponding area under the plasma concentration vs. time curve (AUC) values increased in a dose-related but nonproportional manner for both single (51-1131 micrograms.hr/ml) and multiple (54-858 micrograms.hr/ml) doses. Over the 10-150 mg/kg dosing range, oral plasma clearance and terminal half-life values were found to be 2.4-3.6 ml/min/kg and 2.6-3.7 hr following a single oral administration, and 3.0-4.2 ml/min/kg and 2.0-3.8 hr after multiple doses. There were no significant differences between the pharmacokinetic parameters calculated following the first and fifteenth daily doses of topiramate at the 10 and 40 mg/kg levels, indicating that there was no accumulation and no autoinduction or inhibition of enzymes that metabolize topiramate resulting from multiple dosing at these levels. A slight (24%) decrease in AUC was observed at the 150 mg/kg level after the fifteenth daily dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenicity study of fecapentaene-12 diacetate on skin painting in SENCAR mice.

To investigate the effects of both diol esterification and coadministration with antioxidant on the tumorigenicity of fecapentaene-12 (FP-12) preparations, diacetylfecapentaene-12 (DAFP-12) in dimethylsulfoxide (DMSO) was applied to SENCAR mouse skin with or without the stabilizer, vitamin E, twice/week for 5 weeks, following which all animals were promoted for up to 25 weeks by weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). While positive controls receiving 7,12-dimethylbenz[a]anthracene (DMBA) instead of DAFP-12 in a similar protocol all developed papillomas (average of 23/animal), papilloma incidence in mice given DAFP-12 did not differ significantly from that of the vehicle control. We conclude that DAFP-12 shows little or no tumor initiating activity for mouse skin even when coadministered with vitamin E.

Deuterium isotope effect on the toxicokinetics of monomethylamine in the rat.

The single-dose toxicokinetics of monomethylamine has been characterized in the rat by HPLC assay of serial blood samples. Biphasic first-order elimination was observed following an iv bolus dose of 19 mumol/kg with a terminal half-life of 19.1 +/- 1.3 min (mean +/- SE, N = 4). The apparent steady state volume of distribution, systemic blood clearance, and renal blood clearance were 1.21 +/- 0.09 liter/kg, 53.4 +/- 3.5 ml/min/kg, and 5.72 +/- 0.53 ml/min/kg, respectively. The administration of an intragastric dose permitted the calculation of the systemic bioavailability of monomethylamine as 69 +/- 3%. Duplicate experiments using the structural analogue with deuterium atoms substituted for hydrogens on the methyl group revealed a much slower elimination of the compound, although ultimately, 5 times as much was excreted unchanged in the urine. Isotope effects calculated as the ratios of terminal half-life, systemic blood clearance, and systemic bioavailability were 1.9, 2.2, and 1.8, respectively.

Pharmacokinetics of N-nitrodimethylamine and N-nitromethylamine in the rat.

Oxidative metabolism of radioactively labeled N-nitrodimethylamine in rats was compared with that of N-nitromethylamine. Within 7 h, 20% of N-nitrodimethylamine was metabolized to CO2 but only 4% of N-nitromethylamine. The poor oxidative metabolism of N-nitromethylamine is also reflected in the blood levels determined after i.v. administration to catheterized rats. N-Nitrodimethylamine was cleared rapidly from rat blood, while N-nitromethylamine was rapidly distributed into body water but had a long elimination half-life. An amount equal to 5.2% of the dose of the monomethyl compound was excreted intact in urine, but only 0.004% of the dimethyl compound. The pharmacokinetic data obtained were compared with the published data on the pharmacokinetics of the structural analog N-nitrosodimethylamine.

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Toxicokinetics of N-nitrosodimethylamine in the Syrian golden hamster.

The single-dose toxicokinetics of N-nitrosodimethylamine (NDMA) has been characterized in 8-week-old male Syrian golden hamsters by analysis using high performance liquid chromatography of serial blood samples. An i.v. bolus dose of 4.2 mumols/kg [14C]NDMA revealed biphasic first-order elimination with a terminal half-life of 8.7 +/- 1.0 min (mean +/- SE) for unchanged NDMA and 31.5 +/- 5.5 min for total radioactivity, and evidence for conversion to polar metabolites was seen in the chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NDMA were 51.2 +/- 3.0 ml/min/kg and 582 +/- 60 ml/kg, respectively. No unchanged NDMA was detected in the urine following an i.v. bolus dose of 15 mumols/kg [14C]NDMA, but 31% of the total radioactivity was eliminated by that route. A dose of 38 mumols/kg given by gavage indicated a systemic bioavailability of 11 +/- 4% for unchanged NDMA. Reversible binding of NDMA to hamster plasma proteins was found to be negligible. Estimation of the intrinsic hepatic clearance (ClI) in the hamster produced a value of 648 ml/min/kg, which is greater than that previously obtained for the rat, and indicates that the metabolic capacity of the hamster liver is greater than that of the rat. These results suggest that this difference in ClI may play a role in the previously reported (Lijinsky et al. 1987) switch in organotropism from almost exclusivity for liver tumors in hamsters dosed by gavage to additional high incidences of lung and kidney tumors in the rat.

Single-dose toxicokinetics of N-nitrosomethylethylamine and N-nitrosomethyl (2,2,2-trideuterioethyl)amine in the rat.

To investigate the origins of an organotropic shift toward increasing esophageal carcinogenicity and DNA alkylation caused by beta-trideuteration of the hepatocarcinogen, N-nitrosomethylethylamine (NMEA), the single-dose toxicokinetics of NMEA and N-nitrosomethyl(2,2,2-trideuterioethyl)amine (NMEA-d3) has been characterized in 8-week-old male Fischer 344 rats by analysis using high performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to rats revealed biphasic first order elimination with a terminal half-life of 9.46 +/- 0.69 min for unchanged NMEA and 28.9 +/- 2.4 min for total radioactivity. Extensive conversion to polar metabolites was observed in the chromatograms. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMEA were 39.9 +/- 4.6 ml/min/kg and 496 +/- 36 ml/kg, respectively. There was negligible plasma protein binding and no detectable NMEA was excreted unchanged in the urine. Larger doses given by gavage indicated a systemic bioavailability of 25 +/- 1%. Similar doses of NMEA-d3 given to other groups of rats revealed no significant differences in any of the toxicokinetic parameters. No N-nitrosomethyl(2-hydroxyethyl)amine was found as a detectable metabolite of NMEA or NMEA-d3 in any of the blood or urine samples which were analyzed. When considered together, the data suggest that previously observed differences in organ specificity for the carcinogens, NMEA and NMEA-d3, are not due to differences in the total amounts of nitrosamine reaching particular tissues, but may have other localized causes such as differences in the enzymes responsible for metabolism which are present in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The Fenton degradation as a nonenzymatic model for microsomal denitrosation of N-nitrosodimethylamine.

The microsomal metabolism of the carcinogen N-nitrosodimethylamine (NDMA) was suggested to be initiated by hydrogen atom abstraction to form an alpha-nitrosamino radical, which either oxidizes further to an alpha-hydroxy nitrosamine as the initial product of the activating dealkylation pathway or fragments to the nitric oxide radical and N-methylformaldimine as the first step of the presumably inactivating denitrosation route. To examine the chemistry of the alpha-nitrosamino radical in a nonenzymatic setting, we exposed NDMA to the Fenton reagent, which is known to be capable of abstracting hydrogen atoms from organic species. The products observed were those expected of a denitrosation model. Solutions containing 13 mM [14C]NDMA, 15 mM FeSO4, 15 mM H2O2, and 7.5 mM H2SO4 were kept at 4-10 degrees C for 1 h and then basified to yield methylamine (3.2 +/- 0.5 mM, mean +/- SD, n = 8), formaldehyde (3.1 +/- 0.9 mM), and unreacted nitrosamine (10.2 +/- 0.7 mM) as the only radioactive species detected, with total nitrate/nitrite also being found at a level of 2.8 +/- 0.5 mM. N-Methylformaldiminium ion was identified as an intermediate. The parallels between these results and those seen in the microsomal reaction support the hypothesis that the alpha-nitrosamino radical is a common intermediate in enzymatic denitrosation versus dealkylation of NDMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations of the N-hydroxylation of 3'-hydroxyacetanilide, a non-hepatotoxic positional isomer of acetaminophen.

The hydroxamic acid of 3'-hydroxyacetanilide (AMAP) was synthesized to test the hypothesis that different reactive metabolites of AMAP and acetaminophen account for similarities in covalent binding of the two positional isomers to hepatic proteins, but for differences in their ability to cause hepatotoxicity. N-OH-AMAP was found to be a relatively stable hydroxamic acid, but it was not detected as a metabolite of AMAP formed in vitro by mouse liver microsomes or in urine of mice administered AMAP. Therefore, metabolites other than N-OH-AMAP must be responsible for covalent binding observed with AMAP to mouse liver proteins.

Decomposition and quality control considerations in biological work with fecapentaene preparations.

Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found to decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed to be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of an all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. The mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella typhimurium tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application at hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.

Sex differences in the single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine in the rat.

The single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine (NMHA) has been characterized in 8-week-old Fischer 344 rats by analysis using high-performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to male rats revealed biphasic first-order elimination with a terminal half-life of 37.4 +/- 1.7 min for unchanged NMHA and 101 +/- 6 min for total radioactivity, and extensive conversion to polar metabolites was seen in the high-performance liquid chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMHA were 13.1 +/- 0.9 ml/min/kg, and 685 +/- 31 ml/kg, respectively. Renal blood clearance and intrinsic hepatic clearance were estimated to be 0.805 +/- 0.024 and 16.7 +/- 2.1 ml/min/kg, respectively. A similar dose given to female rats yielded a terminal half-life for NMHA of 27.2 +/- 1.2 min, a steady-state volume of distribution of 652 +/- 23 ml/kg, and systemic blood, renal blood, and intrinsic hepatic clearances of 16.9 +/- 1.3, 1.45 +/- 0.14, and 22.5 +/- 0.3 ml/min/kg, respectively. The sex differences in terminal half-life and systemic blood, renal blood, and intrinsic hepatic clearances were significant at the P less than 0.05 level. Larger doses given by gavage, which appeared to be completely absorbed from the gut, indicated systemic bioavailabilities for unchanged NMHA of 78 +/- 10% and 69 +/- 1% for male and female rats, respectively. Binding of NMHA to plasma proteins was found to be negligible. Taken together the data allow for the conclusion that the observed sex differences in toxicokinetic parameters are due to differences in the intrinsic hepatic clearance of the compound. This difference in the ability of the liver to metabolize NMHA in vivo correlates with and may contribute to the greater susceptibility of female rats to hepatocarcinogenesis and of male rats to development of tumors in the nasal epithelium following oral exposure to NMHA.

Inactivity of fecapentaene-12 as a rodent carcinogen or tumor initiator.

The possible carcinogenic activity of synthetic fecapentaene-12 (FP-12) was studied in several mammalian test systems: (a) for carcinogenicity by intrarectal instillation in male F344/NCr rats as well as by intrarectal and subcutaneous application in male B6C3F1 mice; (b) for initiation by skin painting in female SENCAR mice followed by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), with 7,12-dimethylbenz[a]anthracene (DMBA) followed by TPA as positive control; (c) in a rat subcutaneous granuloma pouch assay in which mutagenicity was measured by induction of 6-thioguanine (6-TG) resistance and carcinogenicity was determined by induction of subcutaneous tumors in the pouch. There was no significant increase in tumor incidence after 72-78 weeks in test (a), although 2 rats receiving FP-12 intrarectally developed colon polyps. FP-12 did not initiate any skin tumors in test (b), nor did it significantly convert DMBA-initiated papillomas into carcinomas when 8 of the positive control mice were given FP-12 weekly for 10 weeks after 10 weeks on the DMBA-TPA regimen. Although FP-12 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were comparably mutagenic in test (c), FP-12 induced no tumors after more than a year in 133 rats at risk while MNNG induced 7 tumors in 107 rats. These rodent assays provide no evidence that FP-12 is a strong carcinogen, although the possibility remains that it may possess weak carcinogenic activity not revealed by these experiments.

Evidence for the involvement of a nitrenium ion in the covalent binding of nitrofurazone to DNA.

We have shown that the xanthine oxidase-catalyzed anaerobic reduction of nitrofurazone in the presence of added DNA leads to the formation of covalently bound adducts. Further, by systematically decreasing the pH of the reaction mixture, we have demonstrated that generation of the reactive species is facilitated under mildly acidic conditions. From these observations, we conclude that it is the nitrenium ion formed from nitrofurazone which binds to DNA.