In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells.

Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa (marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is understood regarding its potential adverse effects. Recent studies revealed that CBD induced toxicity in the male reproductive system of animal models. In this study, we used TM4, an immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels of a functional marker, Wilms' tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites can inhibit cell proliferation in mouse and human Sertoli cells.

Effect of urinary pH upon the renal toxicity of melamine and cyanuric acid.

In 2007, dietary exposure to "scrap melamine' resulted in the death of a large number of cats and dogs, which was attributed to the formation of melamine cyanurate crystals in their kidneys. In this study, we investigated if changes in urinary pH could diminish the renal toxicity associated with exposure to combinations of melamine and cyanuric acid. Female Sprague-Dawley rats were treated for three days with suspensions of melamine and cyanuric acid at doses that were expected to induce renal toxicity. Dosing was then discontinued and the rats were treated for seven days with drinking water solutions (i.e., ammonium chloride and sodium bicarbonate) that would alter urinary pH. The urinary pH of rats administered ammonium chloride drinking water decreased from pH 6.0-6.2 to pH 5.1-5.2. This was accompanied by a decrease in the incidence of melamine cyanurate crystals in the kidneys and a decrease in the incidence of renal lesions. These data suggest that acidification of urine may help overcome the renal toxicities associated with the formation of melamine cyanurate crystals in the kidney.

In vivo localization and postmortem stability of benzo[a]pyrene-DNA adducts.

DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) play a critical role in the etiology of gastrointestinal tract cancers in humans and other species orally exposed to PAHs. Yet, the precise localization of PAH-DNA adducts in the gastrointestinal tract, and the long-term postmortem PAH-DNA adduct stability are unknown. To address these issues, the following experiment was performed. Mice were injected intraperitoneally with the PAH carcinogen benzo[a]pyrene (BP) and euthanized at 24 h. Tissues were harvested either at euthanasia (0 time), or after 4, 8, 12, 24, 48, and 168 hr (7 days) of storage at 4°C. Portions of mouse tissues were formalin-fixed, paraffin-embedded, and immunohistochemically (IHC) evaluated by incubation with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum and H-scoring. The remaining tissues were frozen, and DNA was extracted and assayed for the r7,t8,t9-trihydroxy-c-10-(N 2 -deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct using two quantitative assays, the BPDE-DNA chemiluminescence immunoassay (CIA), and high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ES-MS/MS). By IHC, which required intact nuclei, BPdG adducts were visualized in forestomach basal cells, which included gastric stem cells, for up to 7 days. In proximal small intestine villus epithelium BPdG adducts were visualized for up to 12 hr. By BPDE-DNA CIA and HPLC-ES-MS/MS, both of which used DNA for analysis and correlated well (P= 0.0001), BPdG adducts were unchanged in small intestine, forestomach, and lung stored at 4°C for up to 7 days postmortem. In addition to localization of BPdG adducts, this study reveals the feasibility of examining PAH-DNA adduct formation in wildlife species living in colder climates. Environ. Mol. Mutagen. 61:216-223, 2020. © 2019 Wiley Periodicals, Inc.

Comparative pharmacokinetic and biodistribution study of two distinct squalene-containing oil-in-water emulsion adjuvants in H5N1 influenza vaccines.

BACKGROUND: In recent years, there has been great interest from academia, industry and government scientists for an increased understanding of the mode of action of vaccine adjuvants to characterize the safety and efficacy of vaccines. In this context, pharmacokinetic (PK) and biodistribution studies are useful for quantifying the concentration of vaccine adjuvants in mechanistically or toxicologically relevant target tissues. METHODS: In this study, we conducted a comparative analysis of the PK and biodistribution profile of radiolabeled squalene for up to 336 h (14 days) after intramuscular injection of mice with adjuvanted H5N1 influenza vaccines. The evaluated adjuvants included an experimental-grade squalene-in-water (SQ/W) emulsion (AddaVax®) and an adjuvant system (AS03®) that contained squalene and α-tocopherol in the oil phase of the emulsion. RESULTS: The half-life of the initial exponential decay from quadriceps muscle was 1.5 h for AS03 versus 12.9 h for AddaVax. At early time points (1-6 h), there was about a 10-fold higher concentration of labeled squalene in draining lymph nodes following AS03 injection compared to AddaVax. The area-under-concentration curve up to 336 h (AUC0-336hr) and peak concentration of squalene in spleen (immune organ) was about 1.7-fold higher following injection of AS03 than AddaVax. The peak systemic tissue concentration of squalene from the two adjuvants, with or without antigen, remained below 1% of injected dose for toxicologically relevant target tissues, such as spinal cord, brain, and kidney. The pharmacokinetics of AS03 was unaffected by the presence of H5N1 antigen. CONCLUSIONS: This study demonstrates a rapid decline of AS03 from the quadriceps muscles of mice as compared to conventional SQ/W emulsion adjuvant, with an increased transfer to mechanistically relevant tissues such as local lymph nodes. Systemic tissue exposure to potential toxicological target tissues was very low.

Low dose assessment of the carcinogenicity of furan in male F344/N Nctr rats in a 2-year gavage study.

Furan is a volatile organic chemical that is a contaminant in many common foods. Furan is hepatocarcinogenic in mice and rats; however, the risk to humans from dietary exposure to furan cannot be estimated accurately because the lowest tested dose of furan in a 2-year bioassay in rats gave nearly a 100% incidence of cholangiocarcinoma. To provide bioassay data that can be used in preparing risk assessments, the carcinogenicity of furan was determined in male F344/N Nctr rats administered 0, 0.02, 0.044, 0.092, 0.2, 0.44, 0.92, and 2 mg furan/kg body weight (BW) by gavage 5 days/week for 2 years. Exposure to furan was associated with the development of malignant mesothelioma on membranes surrounding the epididymis and on the testicular tunics, with the increase being significant at 2 mg furan/kg BW. There was also a dose-related increase in the incidence of mononuclear cell leukemia, with the increase in incidence being significant at 0.092, 0.2, 0.92, and 2 mg furan/kg BW. Dose-related non-neoplastic liver lesions included cholangiofibrosis, mixed cell foci, basophilic foci, biliary tract hyperplasia, oval cell hyperplasia, regenerative hyperplasia, and cytoplasmic vacuolization. The most sensitive non-neoplastic lesion was cholangiofibrosis, the frequency of which increased significantly at 0.2 mg furan/kg BW.

Pharmacokinetics and biodistribution of squalene-containing emulsion adjuvant following intramuscular injection of H5N1 influenza vaccine in mice.

Squalene is a component of oil-in-water emulsion adjuvants developed for potential use in some influenza vaccines. The biodistribution of the squalene-containing emulsion adjuvant (AddaVax™) alone and as part of complete H5N1 vaccine was quantified in mechanistically and toxicologically relevant target tissues up to 336 h (14 days) following injection into quadriceps muscle. At 1 h, about 55% of the intramuscularly injected dose of squalene was detected in the local quadriceps muscles and this decreased to 26% at 48 h. Twenty-four hours after the injection, approximately 5%, 1%, and 0.6% of the injected dose was detected in inguinal fat, draining lymph nodes, and sciatic nerve, respectively. The peak concentration for kidney, brain, spinal cord, bone marrow, and spleen was each less than 1% of the injected dose, and H5N1 antigen did not significantly alter the biodistribution of squalene to these tissues. The area-under-blood-concentration curve (AUC) and peak blood concentration (Cmax) of squalene were slightly higher (20-25%) in the presence of H5N1 antigen. A population pharmacokinetic model-based statistical analysis identified body weight and H5N1 antigen as covariates influencing the clearance of squalene. The results contribute to the body of knowledge informing benefit-risk analyses of squalene-containing emulsion vaccine adjuvants.

Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of pyrrolizidine alkaloid-induced tumorigenicity.

Pyrrolizidine alkaloid-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. The U.S. National Toxicology Program (NTP) classified riddelliine, a tumorigenic pyrrolizidine alkaloid, as "reasonably anticipated to be a human carcinogen" in the NTP 12th Report on Carcinogens in 2011. We previously determined that four DNA adducts were formed in rats dosed with riddelliine. The structures of the four DNA adducts were elucidated as (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine adducts (termed as DHP-dG-3 and DHP-dG-4) as the predominant adducts; and (ii) a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl)dehydrosupinidine adducts (termed as DHP-dA-3 and DHP-dA-4 adducts). In this study, we selected a nontumorigenic pyrrolizidine alkaloid, platyphylliine, a pyrrolizidine alkaloid N-oxide, riddelliine N-oxide, and nine tumorigenic pyrrolizidine alkaloids (riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, heliotrine, clivorine, and senkirkine) for study in animals. Seven of the nine tumorigenic pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, are liver carcinogens. At 8-10 weeks of age, female F344 rats were orally gavaged for 3 consecutive days with 4.5 and 24 µmol/kg body weight test article in 0.5 mL of 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed, livers were removed, and liver DNA was isolated for DNA adduct analysis. DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts were formed in the liver of rats treated with the individual seven hepatocarcinogenic pyrrolizidine alkaloids and riddelliine N-oxide. These DNA adducts were not formed in the liver of rats administered retronecine, the nontumorigenic pyrrolizidine alkaloid, platyphylliine, or vehicle control. These results indicate that this set of DNA adducts, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, is a common biological biomarker of pyrrolizidine alkaloid-induced liver tumor formation. To date, this is the first finding that a set of exogenous DNA adducts are commonly formed from a series of tumorigenic xenobiotics.

Dose-response assessment of nephrotoxicity from a twenty-eight-day combined-exposure to melamine and cyanuric acid in F344 rats.

The adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of cats and dogs in the USA and other countries. In a previous 7-day dietary study in F344 rats, we established a no-observed-adverse-effect level (NOAEL) for a co-exposure to melamine and cyanuric acid of 8.6 mg/kg bw/day of each compound, and a benchmark dose lower confidence limit (BMDL) of 8.4-10.9 mg/kg bw/day of each compound. To ascertain the role played by the duration of exposure, we treated F344 rats for 28 days. Groups of male and female rats were fed diet containing 0 (control), 30, 60, 120, 180, 240, or 360 ppm of both melamine and cyanuric acid. The lowest dose that produced histopathological alterations in the kidney was 120 ppm, versus 229 ppm in the 7-day study. Wet-mount analysis of kidney sections demonstrated the formation of melamine cyanurate spherulites in one male and two female rats at the 60 ppm dose and in one female rat at the 30 ppm dose, establishing a NOAEL of 2.1mg/kg bw/day for males and <2.6 mg/kg bw/day for females, and BMDL values as low as 1.6 mg/kg bw/day for both sexes. These data demonstrate that the length of exposure is an important component in the threshold of toxicity from a co-exposure to these compounds and suggest that the current risk assessments based on exposures to melamine alone may not reflect sufficiently the risk of a co-exposure to melamine and cyanuric acid.

Urinary biomarker detection of melamine- and cyanuric acid-induced kidney injury in rats.

Oral coexposure of rats to melamine (MEL) and cyanuric acid (CYA) results in a dose-dependent increase in the formation of MEL-CYA crystals in the kidney. The aim of this study was to determine if urinary biomarkers of acute kidney injury could be used to noninvasively detect renal damage associated with crystal formation in the kidneys of MEL- and CYA-exposed rats. Urine was obtained on days 0 (predose), 2, 4, 14, and 28 from male and female Fischer 344 rats fed a diet supplemented with 0, 120, 180, or 240 ppm each of MEL and CYA. A number of urinary protein biomarkers (kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, albumin, alpha-GST, GST-Yb1, renal papillary antigen 1 [RPA-1], and clusterin) were measured using a multiplex assay system. The results showed that RPA-1 (distal tubule and collecting duct injury biomarker) was elevated on day 28 at the 120 ppm dose and higher in male rats and at the 180 ppm dose and higher in female rats; however, other urinary protein biomarkers were significantly elevated only at the 240 ppm dose. Significant elevation in blood urea nitrogen and serum creatinine levels, and severe renal damage evidenced by histopathology, were observed after 28 days of exposure to the highest dose, despite the fact that MEL-CYA crystals were observable at the 120 and 180 ppm doses. These data indicate that RPA-1 may serve as a noninvasive urinary biomarker for the detection and monitoring of obstructive nephropathy associated with MEL-CYA exposure.

Tumorigenicity of acrylamide and its metabolite glycidamide in the neonatal mouse bioassay.

Acrylamide is a high-volume industrial chemical, a component of cigarette smoke, and a product formed in certain foods prepared at high temperatures. Previously, we compared the extent of DNA adduct formation and mutations in B6C3F(1) /Tk mice treated neonatally with acrylamide or glycidamide to obtain information concerning the mechanism of acrylamide genotoxicity. We have now examined the tumorigenicity of acrylamide and glycidamide in mice treated neonatally. Male B6C3F(1) mice were injected intraperitoneally on postnatal days 1, 8 and 15 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day and the tumorigenicity was assessed after 1 year. Survival in each of the groups was >87%, there were no differences in body weights among the groups, and the only treatment-related neoplasms involved the liver. The incidence of combined hepatocellular adenoma or carcinoma was 3.8% in the control group, 8.3% in the 0.14 mmol acrylamide and glycidamide per kg body weight groups, 4.2% in the 0.70 mmol acrylamide per kg body weight group and 71.4% in the 0.70 mmol glycidamide per kg body weight group. Analysis of the hepatocellular tumors indicated that the increased incidence observed in mice administered 0.70 mmol glycidamide per kg body weight was associated with A → G and A → T mutations at codon 61 of H-ras. These results, combined with our previous data on DNA adduct formation and mutation induction, suggest that the carcinogenicity of acrylamide is dependent on its metabolism to glycidamide, a pathway that is deficient in neonatal mice.

Pharmacokinetics of melamine and cyanuric acid and their combinations in F344 rats.

The intentional adulteration of pet food with melamine and cyanuric acid has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity in a range of experimental animals, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. Given this mode of action, a good understanding of the pharmacokinetic profiles of melamine and cyanuric acid and their combinations is essential to define properly the risk associated with different exposure scenarios. Previous studies have investigated the individual pharmacokinetic profiles of melamine and cyanuric acid. In this work, we report a comparison between the pharmacokinetic profiles of melamine and cyanuric acid administered individually, administered simultaneously as the individual compounds, and administered as a preformed melamine cyanurate complex. Although the oral coadministration of 1 mg/kg body weight of melamine and cyanuric acid did not alter significantly the pharmacokinetic profiles in relation to those determined upon individual oral administration of each compound, the administration of equal amounts of each triazine as the preformed melamine cyanurate complex significantly altered the pharmacokinetics, with reduced bioavailability of both compounds, lower observed maximum serum concentrations, delayed peak concentrations, and prolonged elimination half lives. These results indicate that in order to estimate properly the combined nephrotoxic potential of melamine and cyanuric acid, the experimental design of toxicological experiments and the evaluation of animal or human exposure scenarios should consider the detailed mode of exposure, with particular emphasis on any possible ex vivo formation of melamine cyanurate.

Benzocaine-induced methemoglobinemia in an acute-exposure rat model.

Tricaine methanesulfonate, a sedative for temporarily immobilizing fish, has a 21-day withdrawal time. Benzocaine has been proposed as an alternative sedative because a withdrawal period may not be required. Since benzocaine is known to induce methemoglobinemia, the potential for orally administered benzocaine to induce methemoglobin was assessed in rats. Sprague-Dawley rats were given a single gavage administration of 64mg benzocaine hydrochloride per kg bw and then euthanized at intervals up to 120min. Plasma levels of benzocaine were relatively low at all times, whereas methemoglobin peaked at 24min. Additional rats were orally gavaged with 0-1024mg benzocaine hydrochloride per kg bw and euthanized after 24min. Plasma levels of benzocaine increased from 0.01µM at 2mg per kg bw to 2.9µM at 1024mg per kg bw. Methemoglobin levels did not differ from controls at doses up to 32mg per kg bw in females and 64mg per kg bw in males, whereupon the value increased to ∼80% at 1024mg per kg bw. These data were used to estimate the potential impact of benzocaine residues in fish and suggest that the consumption of fish treated with benzocaine hydrochloride will not cause methemoglobinemia in humans.

Dose-response assessment of nephrotoxicity from a 7-day combined exposure to melamine and cyanuric acid in F344 rats.

The intentional adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. To determine the dose-response for nephrotoxicity upon coadministration of melamine and cyanuric acid, groups of male and female F344 rats (six animals per sex per group) were fed 0 (control), 7, 23, 69, 229, or 694 ppm of both melamine and cyanuric acid; 1388 ppm melamine; or 1388 ppm cyanuric acid in the diet for 7 days. No toxicity was observed in the rats exposed to the individual compounds, whereas anorexia and a statistically significant increase in blood urea nitrogen and serum creatinine levels was observed in the animals treated with 229 and 694 ppm melamine and cyanuric acid. The kidneys of these animals were grossly enlarged and pale yellow. Large numbers of crystalline structures deposited in the tubules were seen on sections in kidneys from all rats in these treatment groups. No significant changes were detected in the remaining treatment groups exposed to both melamine and cyanuric acid. In the melamine-only treatment group, 5 of 12 rats had scattered crystals present in renal tubules when examined by wet mount. These were not observed by histopathology. The observed adverse effect level (8.6 mg/kg bw [body weight]/day) and benchmark dose modeling data (8.4-10.9 mg/kg bw/day) determined in this study suggest that the tolerable daily intake values derived from studies conducted with melamine alone may underestimate the risk from coexposures to melamine and cyanuric acid.

Identification of metabolite profiles of the catechol-O-methyl transferase inhibitor tolcapone in rat urine using LC/MS-based metabonomics analysis.

The process of drug metabolite identification is extremely important for drug efficacy, safety and pharmacokinetics. The traditional method usually involves using a drug with a radioactive labeled nuclei and/or isolating major drug metabolites by HPLC before applying MS and NMR analyses, which requires trained specialists to handle the radioactive compounds and is time consuming for offline-HPLC separation. A method using mass spectrometry-based metabonomics combined with multivariate statistical analysis was applied to rapidly identify metabolite profiles of tolcapone, a catechol-O-methyl transferase inhibitor for Parkinson's disease treatment. The tolcapone metabolites were identified based on the accurate mass measurement (<3 ppm) and MS(2) mass spectrum. In total, 16 tolcapone metabolites were detected and identified, 6 of which have not been reported previously. Our results indicate that the method has the capability to accelerate the process of identifying drug metabolites, ultimately reduce drug development costs, and make the process safer without requiring a drug with radioactive nuclei. Most importantly, the assay can detect the major and minor drug metabolites in a global view. Furthermore, since tolcapone has been associated with idiosyncratic drug induced liver toxicity the rapid detection of tolcapone-related metabolites can provide mechanistic toxicity information related to drug metabolism and the formation of reactive drug metabolites.

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide.

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1-8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F1/Tk(+/+) mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F1/Tk(+/-) mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine, the major glycidamide-DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide approximately 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1-8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1-8 are due to another mechanism.

The liver toxicity biomarker study: phase I design and preliminary results.

Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.

Effect of short-term exposure to zidovudine (AZT) on the expression of mitochondria-related genes in skeletal muscle of neonatal mice.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the main anti-retroviral drug given to HIV-1-infected pregnant women during pregnancy and to their infants after birth to reduce mother-to-child transmission of the virus. In animal studies, however, a significant mitochondrial morphological damage has been reported in skeletal muscle as a consequence of transplacental or perinatal exposure to AZT. Because proper muscle function is highly dependent on efficient mitochondrial function and information on AZT-induced mitochondrial toxicity during neonatal exposure is limited, we investigated the effect of AZT on the expression of 542 mitochondria-related genes encoded by both nuclear and mitochondrial DNA in the skeletal muscle of infant male and female mice using microarray technology. Animals were treated orally by gavage with AZT at 0, 10, 50, 100, and 200mg/kg body weight/day from postnatal day (PND) 1 through 8 and were sacrificed at 1- and 2-h following the last dose on PND 8. These doses in mice correspond to 0, 1.1, 5.5, 11.0, and 22.0mg/kg AZT in human infants [Center for Drug Evaluation and Research (CDER) 2005. Pharmacology and Toxicology, Guidance for industry. Estimating the maximum safe dose in initial clinical trials for therapeutics in adult healthy volunteers, p. 7. http://www.fda.gov/cder/guidance/index.htm.]. Microarray data were analyzed for effects of time, sex, treatment, and their interactions using a fixed effect linear model. The results showed modest, but significant, dose-related responses in the expression level of genes associated with apoptosis, fatty acid metabolism, mitochondrial DNA maintenance, and various mitochondrial membrane transporters. The transcription levels were not significantly different at both time points and were not sex dependent. The results suggest that changes in expression of mitochondria-related genes in skeletal muscle may be an initial response to short-term AZT exposure in infant mice.

Transplacental drug transfer and frequency of Tk and Hprt lymphocyte mutants and peripheral blood micronuclei in mice treated transplacentally with zidovudine and lamivudine.

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations.

High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts.

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Levels of 4-aminobiphenyl-induced somatic H-ras mutation in mouse liver DNA correlate with potential for liver tumor development.

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.