Pharmacokinetics of oseltamivir phosphate and oseltamivir carboxylate in non-pregnant and pregnant rhesus monkeys.

Oseltamivir is an antiviral drug approved to treat influenza in humans. Although the dosing regimen of this drug is well established for non-pregnant patients, it is not clear if the significant physiological alterations associated with pregnancy affect the pharmacokinetics of oseltamivir and, thus, warrant different dosing regimens to assure efficacy. In this study, we investigated the suitability of rhesus macaques as an animal model for studying oseltamivir pharmacokinetics during all trimesters of pregnancy in comparison to pre-pregnant conditions. Specifically, we compared the pharmacokinetics of oseltamivir and its pharmacologically active metabolite oseltamivir carboxylate in rhesus monkeys after intravenous and nasogastric administration of 2.5 mg oseltamivir phosphate/kg body weight given prior to and during the first, second, and third trimesters of pregnancy. Pregnancy had only a modest effect upon the pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate. Monkeys treated intravenously in the third trimester had a reduction in Vd and CL, compared to non-pregnant monkeys. These changes did not occur in the other two trimesters. Pregnant monkeys treated intravenously had 20-25% decrease in AUC0-∞ of oseltamivir carboxylate and a corresponding increase in Vd and CL. Pregnant monkeys treated nasogastrically with oseltamivir phosphate demonstrated a pattern that recapitulated intravenous dosing. Taken together these data indicate that rhesus monkeys are an acceptable model for studying drug-pregnancy interactions.

p53-competent cells and p53-deficient cells display different susceptibility to oxygen functionalized graphene cytotoxicity and genotoxicity.

Due to the distinctive physical, electrical, and chemical properties of graphene nanomaterials, numerous efforts pursuing graphene-based biomedical and industrial applications are underway. Oxidation of pristine graphene surfaces mitigates its otherwise hydrophobic characteristic thereby improving its biocompatibility and functionality. Yet, the potential widespread use of oxidized graphene derivatives raises concern about adverse impacts on human health. The p53 tumor suppressor protein maintains cellular and genetic stability after toxic exposures. Here, we show that p53 functional status correlates with oxygen functionalized graphene (f-G) cytotoxicity and genotoxicity in vitro. The f-G exposed p53-competent cells, but not p53-deficient cells, initiated G0 /G1 phase cell cycle arrest, suppressed reactive oxygen species, and entered apoptosis. There was p53-dependent f-G genotoxicity evident as increased structural chromosome damage, but not increased gene mutation or chromatin loss. In conclusion, the cytotoxic and genotoxic potential for f-G in exposed cells was dependent on the p53 functional status. These findings have broad implications for the safe and effective implementation of oxidized graphene derivatives into biomedical and industrial applications. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Pubertal delay in male nonhuman primates (Macaca mulatta) treated with methylphenidate.

Juvenile male rhesus monkeys treated with methylphenidate hydrochloride (MPH) to evaluate genetic and behavioral toxicity were observed after 14 mo of treatment to have delayed pubertal progression with impaired testicular descent and reduced testicular volume. Further evaluation of animals dosed orally twice a day with (i) 0.5 mL/kg of vehicle (n = 10), (ii) 0.15 mg/kg of MPH increased to 2.5 mg/kg (low dose, n = 10), or (iii) 1.5 mg/kg of MPH increased to 12.5 mg/kg (high dose, n = 10) for a total of 40 mo revealed that testicular volume was significantly reduced (P < 0.05) at months 15 to 19 and month 27. Testicular descent was significantly delayed (P < 0.05) in the high-dose group. Significantly lower serum testosterone levels were detected in both the low- (P = 0.0017) and high-dose (P = 0.0011) animals through month 33 of treatment. Although serum inhibin B levels were increased overall in low-dose animals (P = 0.0328), differences between groups disappeared by the end of the study. Our findings indicate that MPH administration, beginning before puberty, and which produced clinically relevant blood levels of the drug, impaired pubertal testicular development until ∼5 y of age. It was not possible to resolve whether MPH delayed the initiation of the onset of puberty or reduced the early tempo of the developmental process. Regardless, deficits in testicular volume and hormone secretion disappeared over the 40-mo observation period, suggesting that the impact of MPH on puberty is not permanent.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses.

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Evaluation of p53 genotype on gene expression in the testis, liver, and heart from male C57BL/6 mice.

Our laboratory is conducting experiments designed to characterize the role of p53 in gene expression in the TSG-p53® mouse model. In the study reported here, gene expression levels in tissue derived from the testis, liver, and heart of male, 8-9 week old, p53 wild-type (WT), heterozygous (HET) or knockout (KO) mice were determined utilizing a targeted qPCR 84-gene array. The heart, liver and testis were selected because of the unique function and rate of cell division of each tissue. The genes on the arrays were categorized into three Functional Gene Groups, Apoptosis, Cell-Cycle and DNA Repair. Differences in expression of the functional groups were determined by multivariate analysis of variance (MANOVA) and significant (P < 0.05) differences in their expression were found among the heart, liver and testis. Further, the expression of the Functional Gene Groups in each of these tissues was also significantly affected by p53 genotype. These data indicate that gene expression in unperturbed tissue is influenced by the status of p53 genotype, and relates, at least partially, to the function of the tissue.

The genetic toxicity of methylphenidate: a review of the current literature.

Attention deficit/hyperactivity disorder (ADHD), a common children's behavioral disorder, is characterized by inattention, hyperactivity and impulsivity. The disorder is thought to stem from abnormalities in the catecholamine pathway and the symptoms of the disorder have been successfully treated with methylphenidate (MPH) since the FDA approved the drug in the 1950s. MPH underwent the appropriate safety testing as part of the FDA approval process; however, a publication in 2005 that reported significant increases in cytogenetic damage in the lymphocytes of MPH-treated pediatric patients caused concern for patients and their families, the pharmaceutical industry and regulatory agencies. This communication will review the many studies that were subsequently initiated worldwide to address the genetic safety of MPH in both animal models and human subjects. Animal experiments broadened the study protocols used in the 2005 investigation to include a wider dose-range, a longer treatment period and automated scoring of biological endpoints, where possible, to reduce observer bias. The human subject studies replicated the experimental design used in the 2005 study, but increased the treatment periods and the sizes of the study populations. Neither the laboratory animal nor human subject studies found an increase in any of the measures of genetic damage that were evaluated. Taken together, these new studies are consistent with the original safety evaluation of the FDA and do not support the hypothesis that MPH treatment increases the risk of genetic damage in ADHD patients. Published 2012. This article is a US Government work and is in the public domain in the USA.

Evaluation of mutagenic mode of action in Big Blue mice fed methylphenidate for 24 weeks.

Methylphenidate hydrochloride (MPH), a widely prescribed pediatric drug for attention deficit hyperactivity disorder, induced liver adenocarcinomas in B6C3F1 mice exposed to 500 ppm in feed for 2 years (Dunnick and Hailey (1995) [14]). In order to determine if the induction of liver tumors was by a mutagenic mode of action, groups of male Big Blue (BB) mice (B6C3F1 background) were fed diets containing 50-4000 ppm MPH for 4, 12, or 24 weeks. At sacrifice, the livers were removed and the cII mutant frequency (MF) and spectrum of cII mutations were determined. In addition, the frequencies of micronucleated reticulocytes (MN-RETs) and normochromatic erythrocytes (MN-NCEs) were measured in peripheral blood erythrocytes as was the Hprt MF in splenic lymphocytes. Food consumption and body weight gain/loss were recorded weekly for each animal. The levels of MPH and RA were determined immediately before sacrifice in the serum of mice fed MPH for 24 weeks. A significant loss in body weights (p

Evaluation of Macaca mulatta as a model for genotoxicity studies.

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.

The genetic toxicology of methylphenidate hydrochloride in non-human primates.

The studies presented in this work were designed to evaluate the genetic toxicity of methylphenidate hydrochloride (MPH) in non-human primates (NHP) using a long-term, chronic dosing regimen. Thus, approximately two-year old, male rhesus monkeys of Indian origin were orally exposed to MPH diluted in the electrolyte replenisher, Prang, five days per week over a 20-month period. There were 10 animals per dose group and the doses were (1) control, Prang only, (2) low, 0.15 mg/kg of MPH twice per day increased to 2.5mg/kg twice per day and (3) high, 1.5 mg/kg of MPH twice per day increased to 12.5 mg/kg twice per day. Blood samples were obtained from each animal to determine the base-line serum levels of MPH and the major metabolite of MPH in NHP, ritalinic acid (RA). In addition, the base-line frequency of micronucleated erythrocytes (MN-RETs) by flow cytometry, HPRT mutants by a lymphocyte cloning assay, and chromosome aberrations by FISH painting were determined from peripheral blood samples. Once dosing began, the serum levels of MPH and its major metabolite, RA, were determined monthly. The MN-RET frequency and health parameters (CBC, serum chemistries) were also determined monthly. HPRT mutant and chromosome aberration frequencies were measured every three months. CBC values and serum chemistries, with the exception of alanine amino transferase, were within normal limits over the course of drug exposure. The final plasma levels of MPH were similar to those produced by the pediatric dose of 0.3 microg/ml. No significant increases in the frequencies of MN-RETs, HPRT mutants, or chromosome aberrations were detected in the treated animals compared to the control animals over the 20-month exposure period.

Effect of p53 genotype on gene expression profiles in murine liver.

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the "guardian of the genome". Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53(-/-) and p53(+/-) mice. Six male mice from each genotype (p53(+/+), p53(+/-), and p53(-/-)) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53(+/+) and p53(+/-) or between p53(+/+) and p53(-/-) at the level of p < or = 0.05. Both genes with increased expression and decreased expression were identified in p53(+/-) and in p53(-/-) mice. Most notable in the gene list derived from the p53(+/-) mice was the significant reduction in p53 mRNA. In the p53(-/-) mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.

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.

Whole genome and normalized mRNA sequencing reveal genetic status of TK6, WTK1, and NH32 human B-lymphoblastoid cell lines.

Closely related TK6, WTK1, and NH32 human B-lymphoblastoid cell lines differ in their p53 functional status. These lines are used frequently in genotoxicity studies and in studies aimed at understanding the role of p53 in DNA repair. Despite their routine use, little is known about the genetic status of these cells. To provide insight into their genetic composition, we sequenced and analyzed the entire genome of TK6 cells, as well as the normalized transcriptomes of TK6, WTK1, and NH32 cells. Whole genome sequencing (WGS) identified 21,561 genes and 5.17×10(6) small variants. Within the small variants, 50.54% were naturally occurring single nucleotide polymorphisms (SNPs) and 49.46% were mutations. The mutations were comprised of 92.97% single base-pair substitutions and 7.03% insertions or deletions (indels). The number of predicted genes, SNPs, and small mutations are similar to frequencies observed in the human population in general. Normalized mRNA-seq analysis identified the expression of transcripts bearing SNPs or mutations for TK6, WTK1, and NH32 as 2.88%, 2.04%, and 1.71%, respectively, and several of the variant transcripts identified appear to have important implications in genetic toxicology. These include a single base deletion mutation in the ferritin heavy chain gene (FTH1) resulting in a frame shift and protein truncation in TK6 that impairs iron metabolism. SNPs in the thiopurine S-methyltransferase (TPMT) gene (TPMT*3A SNP), and in the xenobiotic metabolizing enzyme, NADPH quinine oxidoreductase 1 (NQO1) gene (NQO1*2 SNP), are both associated with decreased enzyme activity. The clinically relevant TPMT*3A and NQO1*2 SNPs can make these cell lines useful in pharmacogenetic studies aimed at improving or tailoring drug treatment regimens that minimize toxicity and enhance efficacy.

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells.

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice.

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5'-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk+/+, Tk+/- and Tk-/- mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk+/+ and Tk+/- mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk-/- mice. Unexpectedly, neonatal Tk-/- mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk+/+ and Tk+/- mice (e.g., approximately 3.4% MN-RETs and approximately 4.8% MN-NCEs in Tk-/- mice versus approximately 0.7 and approximately 0.6% MN-RETs and MN-NCEs in neonatal Tk+/+ mice). Additional assays performed on untreated Tk-/- mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk-/- mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk-/- mice suggest the presence of an endogenous genotoxic activity in these mice.

Dye bias correction in dual-labeled cDNA microarray gene expression measurements.

A significant limitation to the analytical accuracy and precision of dual-labeled spotted cDNA microarrays is the signal error due to dye bias. Transcript-dependent dye bias may be due to gene-specific differences of incorporation of two distinctly different chemical dyes and the resultant differential hybridization efficiencies of these two chemically different targets for the same probe. Several approaches were used to assess and minimize the effects of dye bias on fluorescent hybridization signals and maximize the experimental design efficiency of a cell culture experiment. Dye bias was measured at the individual transcript level within each batch of simultaneously processed arrays by replicate dual-labeled split-control sample hybridizations and accounted for a significant component of fluorescent signal differences. This transcript-dependent dye bias alone could introduce unacceptably high numbers of both false-positive and false-negative signals. We found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and therefore measurable and correctable. The additional microarrays and reagents required for paired technical replicate dye-swap corrections commonly performed to control for dye bias could be costly to end users. Incorporating split-control microarrays within a set of concurrently processed hybridizations to specifically measure dye bias can eliminate the need for technical dye swap replicates and reduce microarray and reagent costs while maintaining experimental accuracy and technical precision. These data support a practical and more efficient experimental design to measure and mathematically correct for dye bias.

A role for p53 in the frequency and mechanism of mutation.

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.

Detection of mutation in transgenic CHO cells using green fluorescent protein as a reporter.

A novel approach was developed for rapidly estimating the frequency of specific mutations in genetically engineered Chinese hamster ovary (CHO) cells. We designed double-transgenic CHO cell lines that contain a transgene consisting of the sequence coding for green fluorescent protein under the control of a tetracycline (Tet) responsive promoter and a second transgene coding for the constitutively expressed Tet repressor. Cultures of these CHO cells were treated with gamma-radiation, N-methyl-N-nitrosourea or methyl methanesulfonate, and the fluorescence of individual cells from both control and treated cultures was measured by flow cytometry. The treatments increased the number of highly fluorescent cells, those with presumed mutations in the Tet-repressor gene. Mutant cells from gamma-radiation-exposed cultures were isolated by fluorescence-activated cell sorting, cultured, and individual clones expanded. A PCR-based analysis indicated that the highly fluorescent expanded cells had lost the transgene coding for the Tet repressor, suggesting that the system mainly detects large genetic alterations. A similar approach may be useful for making high-throughput in vivo models for mutation detection.

Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice.

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.

Development of a physiologically based model to describe the pharmacokinetics of methylphenidate in juvenile and adult humans and nonhuman primates.

The widespread usage of methylphenidate (MPH) in the pediatric population has received considerable attention due to its potential effect on child development. For the first time a physiologically based pharmacokinetic (PBPK) model has been developed in juvenile and adult humans and nonhuman primates to quantitatively evaluate species- and age-dependent enantiomer specific pharmacokinetics of MPH and its primary metabolite ritalinic acid. The PBPK model was first calibrated in adult humans using in vitro enzyme kinetic data of MPH enantiomers, together with plasma and urine pharmacokinetic data with MPH in adult humans. Metabolism of MPH in the small intestine was assumed to account for the low oral bioavailability of MPH. Due to lack of information, model development for children and juvenile and adult nonhuman primates primarily relied on intra- and interspecies extrapolation using allometric scaling. The juvenile monkeys appear to metabolize MPH more rapidly than adult monkeys and humans, both adults and children. Model prediction performance is comparable between juvenile monkeys and children, with average root mean squared error values of 4.1 and 2.1, providing scientific basis for interspecies extrapolation of toxicity findings. Model estimated human equivalent doses in children that achieve similar internal dose metrics to those associated with pubertal delays in juvenile monkeys were found to be close to the therapeutic doses of MPH used in pediatric patients. This computational analysis suggests that continued pharmacovigilance assessment is prudent for the safe use of MPH.

Pharmacokinetics, dose-range, and mutagenicity studies of methylphenidate hydrochloride in B6C3F1 mice.

Methylphenidate hydrochloride (MPH) is one of the most frequently prescribed pediatric drugs for the treatment of attention deficit hyperactivity disorder. In a recent study, increased hepatic adenomas were observed in B6C3F1 mice treated with MPH in their diet. To evaluate the reactive metabolite, ritalinic acid (RA) of MPH and its mode of action in mice, we conducted extensive investigations on the pharmacokinetics (PK) and genotoxicity of the drug in B6C3F1 mice. For the PK study, male B6C3F1 mice were gavaged once with 3 mg/kg body weight (BW) of MPH and groups of mice were sacrificed at various time points (0.25-24 hr) for serum analysis of MPH and RA concentrations. Groups of male B6C3F1 mice were fed diets containing 0, 250, 500, 1,000, 2,000, or 4,000 ppm of MPH for 28 days to determine the appropriate doses for 24-week transgenic mutation studies. Also, the micronucleus frequencies (MN-RETs and MN-NCEs), and the lymphocyte Hprt mutants were determined in peripheral blood and splenic lymphocytes, respectively. Mice fed 4,000 ppm of MPH lost significant BW compared to control mice (P < 0.01). There was a significant increase in the average liver weights whereas kidneys, seminal vesicle, testes, thymus, and urinary bladder weights of mice fed higher doses of MPH were significantly lower than the control group (P < or = 0.05). There was no significant increase in either the Hprt mutant frequency or the micronucleus frequency in the treated animals. These results indicated that although MPH induced liver hypertrophy in mice, no genotoxicity was observed.