Identification of pyrroloformamide as a cytokinesis modulator.

Discovered in the late 1940s, the pyrrolinonodithioles represent a family of potent disulfide-containing natural products. Although they are understood in a synthetic and biosynthetic context, the biological role of these materials remains unresolved. To date, their activity has been suggested to arise through regulating RNA metabolism, and more recently they have been suggested to function as backup thiols for detoxification. Using materials identified through a natural products program, we now identify the biological function of one member of this family, pyrroloformamide, as an antimitotic agent acting, in part, by disrupting cytokinesis.

Cytotoxic tetraprenylated alkaloids from the South China Sea gorgonian Euplexaura robusta.

Nine achiral tetraprenylated alkaloids, including three new compounds, named malonganenones I-K (1-3, resp.), together with six known analogs, 4-9, were isolated from the gorgonian Euplexaura robusta collected from Weizhou Island of Guangxi Province, China. The structures of compounds 1-3 were elucidated by extensive spectral analyses, especially of their 1D- and 2D-NMR data. Compounds 1, 4, 6, and 7 showed moderate cytotoxicities against K562 and HeLa tumor cell lines with IC(50) values ranging from 0.35 to 10.82 µM. Compound 6 also showed moderate inhibitory activity against c-Met kinase at a concentration of 10 µM.

Liver and heart toxicity due to 90-day oral exposure of ICR mice to N,N-dimethylformamide.

N,N-dimethylformamide (DMF) is a colorless liquid with a faint amine odor, which is widely used in the world. DMF exposure may induce adverse effects on liver, but few studies showed damage to heart after exposure to DMF. In the present study, DMF was administered to ICR mice with the doses of 0.32, 0.63 and 1.26 g/kg of body weight by gavage for 90 days. The increase in the relative liver weight is accompanied with the presence of the centrilobular hepatocellular hypertrophy as well as increased serum levels of aspartate transaminase (AST) and alanine transaminase (ALT). An increase of malondialdehyde (MDA) level was shown in liver homogenate, while superoxide dismutase (SOD) and glutathione (GSH) activities decreased. Heart damage was also shown in mice exposed to DMF for 90 days, although pathological examination showed only slight inflammatory cell infiltration. Increased levels of serum lactate dehydrogenase (LDH), isoenzymes of creatine kinase (CK-MB) and cardiac troponin I (cTnI) were shown. Increased level of MDA was also shown in heart homogenate, in contrast with the decreased activity of SOD. These data suggested that the administration of DMF could induce liver and heart injuries and oxidative stress was involved in the toxic effects.

Role of urinary biomarkers of N,N-dimethylformamide in the early detection of hepatic injury among occupational exposed workers.

AIM: To determine the sensitive and convenient biomarkers for the early detection of hepatic injury in N,N-dimethylformamide (DMF) exposed workers. METHODS: Seventy-nine individuals in a synthetic leather factory were investigated with questionnaire survey. The air samples, urine samples, and blood samples were collected at the specific time point. Airborne DMF and the urine metabolites of DMF were measured by gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Traditional liver function tests and hepatic fibrosis parameters were performed by auto-chemistry analyzer and ELISA methods. RESULTS: The urine concentration of N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), one of the metabolites of DMF, was positively correlated with activities of liver function enzymes. About 60% subjects with urine AMCC concentration above 40 mg/g creatinine showed raised liver enzymes activities. In terms of hepatic fibrosis parameters, we found 4 of 5 abnormal total serum bile acid (SBA) and 4 of 4 abnormal serum hyaluronidase (HA) among workers with higher amount of urine AMCC. CONCLUSION: Workers exposed to DMF with higher urine AMCC levels were more likely to develop liver diseases. In addition, SBA and HA have the potential to act as early indicators of toxic hepatic fibrosis activities for occupational health surveillance.

Synergistic hepatotoxicity of N,N-dimethylformamide with carbon tetrachloride in association with endoplasmic reticulum stress.

N,N-Dimethylformamide (DMF) is an organic solvent extensively used in industries such as synthetic leather, fibers and films, and induces liver toxicity and carcinogenesis. Despite a series of experimental and clinical reports on DMF-induced liver failure, the mechanism of toxicity is yet unclear. This study investigated whether DMF in combination with a low dose of hepatotoxicant enhances hepatotoxicity, and if so, on what mechanistic basis. Treatment of rats with either DMF (50-500mg/kg/day, for 3 days) or a single low dose of CCl(4) (0.2ml/kg) alone caused small increases in plasma transaminases and lactate dehydrogenase activities. However, combinatorial treatment of DMF with CCl(4) markedly increased blood biochemical changes. Histopathology confirmed the synergism in hepatotoxicity. Moreover, DMF+CCl(4) caused PARP cleavage and caspase-3 activation, but decreased the level of Bcl-xL, all of which confirmed apoptosis of hepatocytes. Consistently, DMF+CCl(4) treatment markedly increased lipid peroxidation. By contrast, treatment of DMF in combination with lipopolysaccharide, acetaminophen or d-galactosamine caused no enhanced hepatotoxicity. Given the link between endoplasmic reticulum (ER) dysfunction and cell death, ER stress response was monitored after DMF and/or CCl(4) treatment. Whereas either DMF or CCl(4) treatment alone marginally changed the expression levels of glucose-regulated protein 78 and 94 and phosphorylated PKR-like ER-localized eIF2alpha kinase, concomitant treatment with DMF and CCl(4) synergistically induced them with increases in glucose-regulated protein 78 and C/EBP homologous protein mRNAs. Our results demonstrate that DMF treatment in combination with CCl(4) synergistically increases hepatocyte death, which may be associated with the induction of severe ER stress.

Copper(II) complexes with 2-pyridineformamide-derived thiosemicarbazones: spectral studies and toxicity against Artemia salina.

The copper(II) complexes [Cu(H2Am4DH)Cl2] (1), [Cu(H2Am4Me)Cl2] (2), [Cu(H2Am4Et)Cl2] (3) and [Cu(2Am4Ph)Cl] (4) with 2-pyridineformamide thiosemicarbazone (H2Am4DH) and its N(4)-methyl (H2Am4Me), N(4)-ethyl (H2Am4Et) and N(4)-phenyl (H2Am4Ph) derivatives were studied by means of infrared and EPR spectral techniques. The crystal structure of 4 was determined. The studied compounds proved to be toxic to Artemia salina, suggesting that they could present cytotoxic activity against solid tumors. Among the free thiosemicarbazones H2Am4Ph presented higher toxicity than all other compounds, which showed comparable effects. In the case of complexes 2 and 3 toxicity is probably attributable to the complex as an entity or to a synergistic effect involving the thiosemicarbazone and copper. H2Am4Ph and complexes 2 and 3 revealed to be the most promising compounds as potential antineoplasic agents.

Enhanced hepatocarcinogenicity by combined inhalation and oral exposures to N,N-dimethylformamide in male rats.

N,N-Dimethylformamide (DMF), a ubiquitous contaminant in living and working environments, enters the human body by inhalation, as well as by oral and dermal routes of exposure. In order to provide bioassay data for carcinogenic risk assessment of humans exposed to DMF by multiple routes of exposure, hepatocarcinogenic effect of combined inhalation and oral exposures of rats to DMF was examined. A group of 50 male F344 rats, 6-week-old, was exposed by inhalation to 0 (clean air), 200, or 400 ppm (v/v) of DMF vapor-containing air for 6 hr/day and 5 days/week during a 104-week period, and each inhalation group was given ad libitum DMF-formulated drinking water at 0, 800 or 1,600 ppm (w/w) for 104 weeks. Incidences of hepatocellular adenomas and carcinomas and their combined incidences were significantly increased in the combined-exposure groups compared with the untreated control group or each of the inhalation-alone and oral-alone groups with matching concentrations. Incidences of hepatocellular adenomas and carcinomas induced by the combined exposures were greater than the sum of the two incidences of the hepatocellular adenomas and carcinomas induced by the single-route exposures through inhalation and ingestion. The combined exposures enhanced tumor malignancy. It was concluded that the combined inhalation and oral exposures markedly enhance the incidences and malignancy of hepatocellular tumors, suggesting that the hepatocarcinogenic effect of the combined exposures is greater than the effect that would be expected under the assumption that the two effects of single-route exposures through inhalation and drinking are additive.

Toxicology and carcinogenesis studies of formamide (Cas No. 75-12-7) in F344/N rats and B6C3F1 mice (gavage studies).

UNLABELLED: Formamide is used as a softener for paper, gums, and animal glues; as an ionizing solvent; and in the manufacture of formic esters and hydrocyanic acid. Formamide was nominated for reproductive and genetic toxicity evaluation by the Environmental Defense Fund and for carcinogenicity evaluation by the National Cancer Institute because of the potential for human exposure associated with its widespread industrial use, the absence of data adequately characterizing its potential for reproductive and genetic toxicity, and the fact that acetamide, a compound structurally related to form-amide, is hepatocarcinogenic in rats when administered in feed. Male and female F344/N rats and B6C3F1 mice were administered formamide (approximately 100% pure) in deionized water by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, Drosophila melanogaster, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 10, 20, 40, 80, or 160 mg formamide/kg body weight in deionized water by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) and five male and five female rats (plasma concentration study) were administered the same doses, 5 days per week for up to 14 weeks. All core study rats survived to the end of the study. Mean body weights of females in the 40 mg/kg group and males and females in the 80 and 160 mg/kg groups were significantly less than those of the vehicle controls. On day 23 and at week 14, there was a dose-related increase in the erythron, evidenced by increases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. The incidences of degeneration of the germinal epithelium of the testes and epididymis were significantly increased in 160 mg/kg males. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 10, 20, 40, 80, or 160 mg formamide/kg body weight in deionized water by gavage, 5 days per week for 14 weeks. Additional groups of five male and five female mice (plasma concentration study) were administered the same doses, 5 days per week for 14 weeks. All mice survived to the end of the study. Final mean body weights of the 80 and 160 mg/kg males and mean body weight gains of 40, 80, and 160 mg/kg males were significantly less than those of the vehicle controls. Dosed females differed significantly from vehicle controls in the relative amount of time spent in the estrous stages. All 160 mg/kg males had abnormal residual bodies in the testes. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 20, 40, or 80 mg formamide/kg body weight, 5 days per week for 104 to 105 weeks in deionized water by gavage. Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 80 mg/kg males were less than those of the vehicle controls throughout most of the study. Mean body weights of 40 and 80 mg/kg females were somewhat less than those of the vehicle controls during the second year of the study. A significant increase in the incidence of bone marrow hyperplasia occurred in 80 mg/kg males. No neoplasms were attributed to exposure to formamide. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 20, 40, or 80 mg formamide/kg body weight, 5 days per week for 104 to 105 weeks in deionized water by gavage. Survival of all dosed groups of mice was similar to that of the vehicle controls. Mean body weights of 80 mg/kg males and females were generally less than those of the vehicle controls throughout the study; mean body weights of 40 mg/kg females were generally less after week 13 of the study. The incidences of hemangiosarcoma of the liver occurred with a positive trend in males, and the incidences were significantly increased in the 40 and 80 mg/kg groups. The incidence of hepatocellular adenoma or carcinoma (combined) in 80 mg/kg females was significantly increased. The incidences of mineralization of the testicular arteries and testicular tunic were significantly increased in 80 mg/kg males. The incidence of hematopoietic cell proliferation of the spleen was significantly increased in 80 mg/kg males. GENETIC TOXICOLOGY: Formamide gave no evidence for mutagenicity in a series of short-term assays. In three independent Ames assays, formamide was not mutagenic in any of several strains of S. typhimurium tested with and without rat or hamster liver S9 activation enzymes or in E. coli strain WP uvrA pKM101 tested with and without 10% rat liver S9. Negative results were obtained in a test for induction of sex-linked recessive lethal mutations in germ cells of male D. melanogaster treated with formamide either by feeding or injection. Formamide did not induce increases in micronucleated erythrocytes in male or female mice treated by gavage for 3 months. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of form-amide in male or female F344/N rats administered 20, 40, or 80 mg/kg. There was clear evidence of carcinogenic activity of formamide in male B6C3F1 mice based on increased incidences of hemangiosarcoma of the liver. There was equivocal evidence of carcinogenic activity of formamide in female B6C3F1 mice based on increased incidences of hepatocellular adenoma or carcinoma (combined). An increased incidence of bone marrow hyperplasia occurred in male rats. Mineralization of the testicular arteries and tunic and hematopoietic cell proliferation of the spleen in male mice were also associated with administration of formamide.

Mitochondrial DNA alterations in blood of the humans exposed to N,N-dimethylformamide.

N,N-Dimethylformamide (DMF) has been widely used in industries because of its extensive miscibility with water and solvents. Its health effects include hepatotoxicity and male reproductoxicity, possibly linked with mitochondrial DNA (mtDNA) alterations including mtDNA common deletion (DeltamtDNA(4977)) and mtDNA copy number. The relationship between DMF exposure and mtDNA alterations, however, has not been postulated yet. The purposes of this study were to investigate whether the DMF exposure is associated with DeltamtDNA(4977) and mtDNA copy number and to evaluate the DMF-derived mtDNA alterations are more associated with exposure to the airborne DMF concentrations or to the levels of two urinary DMF biomarkers of N-methylformamide (NMF) and N-acetyl-S-(N-methylcarbamoryl) cysteine(AMCC). Thirteen DMF-exposed workers and 13 age and seniority-matched control workers in a synthetic leather factory were monitored on their airborne DMF, NMF and AMCC in the urine as well as DeltamtDNA(4977) and mtDNA copy number in blood cells. We found that the frequencies of relative DeltamtDNA(4977) in DMF-exposed group were significantly higher than those in the control group. Moreover, elevation in the proportion of DeltamtDNA(4977) of individuals with high urine AMCC (U-AMCC) and airborne DMF levels were significantly higher than those without. We conclude that long-term exposure to DMF is highly associated with the alterations of mtDNA in urine and blood cells. The DeltamtDNA(4977) was more significantly related to repeated exposure to DMF and mtDNA copy number was more closely related to short-term DMF exposure. We also confirmed that U-AMCC is more appropriate to serve as a toxicity biomarker for DMF exposure than U-NMF. Further study with a larger number of subjects is warranted.

Studies on N4-(2-deoxy-D-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine (Fapy.dA) and N6-(2-deoxy-D-pentofuranosyl)-6-diamino-5-formamido-4-hydroxypyrimidine (Fapy.dG).

Exposure of DNA to oxidative stress produces a variety of DNA lesions including the formamidopyrimidines, which are derived from the purines. These lesions may play important roles in carcinogenesis. We achieved the first chemical syntheses of a monomeric form of Fapy-dA (1) and oligonucleotides containing this lesion or Fapy-dG at a defined site. Monomeric Fapy-dA readily epimerized at 25 degrees C in phosphate buffer (pH 7.5). The beta-anomer was favored by a ratio of 1.33:1.0, and equilibration was achieved in less than 7 h. Deglycosylation of Fapy-dA in the monomer follows first-order kinetics from 37 to 90 degrees C. The rate constants for deglycosylation of Fapy-dA in the monomeric and oligonucleotide substrates were measured at a common temperature (55 degrees C) and found to be the same within experimental error (t(1/2) = 20.5 h). Implementation of the activation parameters measured for the deglycosylation of 1 indicates that the half-life for deglycosylation of Fapy-dA at 37 degrees C is approximately 103 h. Analysis of the rate constant for deglycosylation of Fapy-dG in an oligonucleotide, revealed that this lesion is approximately 25 times more resistant to hydrolysis than Fapy-dA at 55 degrees C. These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in DNA so as to warrant investigation of their genotoxicity, and both anomers will be present during this time.

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides.

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.

Biological monitoring of workers exposed to N,N-dimethylformamide in the synthetic fibre industry.

OBJECTIVES: Monitoring of workplace air and biological monitoring of 23 workers exposed to N,N-dimethylformamide (DMF) in the polyacrylic fibre industry was carried out on 4 consecutive days. The main focus of the investigation was to study the relationship between external and internal exposure, the suitability of the metabolites of DMF for biological monitoring and their toxicokinetic behaviour in humans. METHODS: Air samples were collected using personal air samplers. The limit of detection (LOD) for DMF using an analytical method recommended by the Deutsche Forschungsgemeinschaft (DFG) was 0.1 ppm. The urinary metabolites, N-hydroxymethyl-N-methylformamide (HMMF), N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), were determined in one analytical run by gas chromatography with thermionic sensitive detection (GC/TSD). The total sum of HMMF and NMF was determined in the form of NMF. The LOD was 1.0 mg/l for NMF and 0.5 mg/l for AMCC. RESULTS AND CONCLUSIONS: The external exposure to DMF vapour varied greatly depending on the workplace (median 1.74 ppm, range < 0.1-159.77 ppm). Urinary NMF concentrations were highest in post-shift samples. They also covered a wide range (< 1.0-108.7 mg/l). This variation was probably the result of different concentrations of DMF in the air at different workplaces, dermal absorption and differences in the protective measures implemented by each individual (gloves, gas masks etc.). The urinary NMF concentrations had decreased almost to zero by the beginning of the next shift. The median half-time for NMF was determined to be 5.1 h. The concentrations of AMCC in urine were determined to be in the range from < 0.5 to 204.9 mg/l. Unlike the concentrations of NMF, the AMCC concentrations did not decrease during the intervals between the shifts. For the exposure situation investigated in our study, a steady state was found between the external exposure to DMF and the levels of AMCC excreted in urine about 2 days after the beginning of exposure. AMCC is therefore excreted more slowly than NMF. The half-time for AMCC is more than 16 h. Linear regression analysis for external exposure and urinary excretion of metabolites was carried out for a sub-group of 12 workers. External exposure to 10 ppm DMF in air (the current German MAK value) corresponds to an average NMF concentration of about 27.9 mg/l in post-shift urine from the same day and an average AMCC concentration of 69.2 mg/l in pre-shift urine from the following day. NMF in urine samples therefore represents an index of daily exposure to DMF, while AMCC represents an index of the average exposure over the preceding working days. AMCC is considered to be better suited for biomonitoring purposes because (1) it has a longer half-time than NMF and (2) its formation in humans is more closely related to DMF toxicity.

Nephrotoxicity of thioformamide, a proximate toxicant of nephrotoxic thiazoles, in mice depleted of glutathione.

Administration of thioformamide (TFA) (> 2 mmol/kg, sc) in combination with DL-buthionine sulfoximine (BSO) (4 mmol/kg, i.p.), an inhibitor of glutathione (GSH) synthesis, caused kidney injury in male mice. The injury was characterized by tubular necrosis, increases in relative kidney weight and serum urea nitrogen concentration, and a decrease in renal GSH concentration. In contrast to results in male mice, no overt nephrotoxic effects were observed in female mice given TFA in combination with BSO. These features of nephrotoxicity were very similar to those reported for thiabendazole and other nephrotoxic thiazoles in mice depleted of GSH by treatment with BSO; this resemblance supports the suggestion that TFA as a ring cleavage metabolite, or a further metabolite thereof, is responsible for the toxicity of the nephrotoxic thiazoles (Mizutani, T., Yoshida, K., and Kawazoe, S. (1993) Chem. Res. Toxicol. 6, 174-179).

Developmental toxicity of inhaled N-methylformamide in the rat.

The developmental toxicity of N-methylformamide (MMF), an industrial chemical intermediate used in the production of agrichemicals, was examined in pregnant rats. MMF was administered by nose-only inhalation, 6 hr daily on Days 7-16 of gestation (the day copulation was confirmed was termed Day 1 of gestation, Day 1G) at exposure concentrations of 0, 15, 50, or 150 ppm. Dams were regularly monitored throughout gestation for body weight gain, feed consumption, and clinical signs. Cesarean sections were performed on Day 22G and the offspring were examined. Maternal toxicity was evident in dams exposed to 50 or 150 ppm; one dam exposed to 150 ppm died on Day 14G (considered to be treatment-related) and dams in the 50 and 150 ppm groups exhibited concentration-related clinical findings. Clinical signs of wheezing and rattling were observed both during and after the exposure period. The 150 ppm group also showed significant decreases in weight gain and feed consumption. A significant increase in the mean number of resorptions per litter at the 150 ppm level indicated an embryolethal effect. Developmental toxicity was apparent by a significant decrease in mean fetal body weight and increases in fetal malformations (subcutaneous cysts on the head, microphthalmia, anophthalmia, fused ribs and/or vertebra, and distended brain ventricles) and variations (misaligned and fused sternebrae) due to retarded development at 150 ppm. Significant fetal body weight decreases were also present at 50 ppm. Thus, in this study, the no-observable-adverse-effect level for both dam and fetus was 15 ppm MMF, indicating that for the parameters included in this study, the conceptus is not uniquely sensitive to MMF.

Developmental toxicity of N-methylformamide administered by gavage to CD rats and New Zealand white rabbits.

N-Methylformamide (NMF) is a metabolite of dimethylformamide (DMF), a solvent with wide applications in the chemical industry. The potential developmental toxicity of NMF was evaluated in CD rats and New Zealand white rabbits. Pregnant rats and rabbits were dosed once daily by gavage on Gestation Days 6-15 and 6-18, respectively. Doses for rats were 0, 1, 5, 10, or 75 mg/kg; doses for rabbits were 0, 5, 10, or 50 mg/kg. Cesarean sections were performed on rats and rabbits on Gestation Days 20 and 29, respectively. No treatment-related maternal deaths or clinical signs occurred in either species. Body weight gain and food consumption were depressed in rats given 75 mg/kg and rabbits given 50 mg/kg. Fetal viability was reduced at 75 mg/kg in rats and at 50 mg/kg in rabbits. In rats, a significant increase in the incidence of malformations including cephalocele and sternoschisis was observed in fetuses from the 75 mg/kg group. In addition, a developmental delay was indicated by reduction of fetal weight and by a significant increase in the occurrence of incomplete ossification of various skeletal structures. In the rabbit, fetal body weight was reduced at 50 mg/kg. Malformations observed at 50 mg/kg included gastroschisis, cephalocele, domed head, flexed paw, and skull and sternum anomalies. The lowest-observed-adverse-effect levels for maternal and developmental toxicity in the rat and rabbit were 75 and 50 mg/kg, respectively. The no-observed-adverse-effect level for maternal and developmental toxicity in the rat and rabbit was 10 mg/kg.

Pre-treatment of human osteosarcoma cells with N-methylformamide enhances P-glycoprotein expression and resistance to doxorubicin.

N-methylformamide (NMF), a powerful differentiating agent, has been extensively used in experimental and preclinical cancer chemotherapy studies, alone or in association with conventional anti-cancer drugs. To evaluate the use of this molecule in the treatment of osteosarcoma (OS), we have analyzed the effects of NMF and doxorubicin (DXR) on DXR-sensitive and -resistant human OS cell lines. Our study shows that NMF exerts remarkable effects on cell proliferation and, in Saos-2 and SARG cells, also induces differentiation, as shown by increasing alkaline phosphatase activity. Moreover, NMF increases the cytotoxic activity of DXR when administered after the drug, in both DXR-sensitive and -resistant cells. However, when this agent is given before DXR, it enhances P-glycoprotein expression in U-2 OS cell lines. This over-expression is associated with reduced DXR accumulation within cells and with significant enhancement of resistance to DXR.

Possible role of thioformamide as a proximate toxicant in the nephrotoxicity of thiabendazole and related thiazoles in glutathione-depleted mice: structure-toxicity and metabolic studies.

In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), thiabendazole (TBZ) causes renal injury characterized by an increase in serum urea nitrogen (SUN) concentration and by tubular necrosis. Previous studies have shown that TBZ requires metabolic activation before it produces nephrotoxicity and that the structure contributing to the toxicity of TBZ is the thiazole moiety of the molecule. TBZ and its thiazole analogues were examined for the ability to increase SUN concentration and serum alanine aminotransferase activity in GSH-depleted mice. Unsubstituted thiazole and thiazoles with 4- and/or 5-, and no 2-, substituents caused marked increases in SUN concentration, suggesting nephrotoxicity. Furthermore, the nephrotoxic potency of these thiazoles decreased with the increasing number and bulk of the 4- and/or 5-substituents. On the other hand, the target organ (the kidney or liver) and the toxic potency of 4-methylthiazoles were markedly altered with the type of substituents at the 2-position. These observations and the known toxicity of thiono-sulfur compounds led us to the hypothesis that the nephrotoxic thiazoles, which lack 2-substituents, would undergo microsomal epoxidation of the C-4,5 double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form thioformamide, a possibly toxic metabolite. Evidence for this hypothesis was provided by the results that thioformamide and tert-butylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 4-tert-butylthiazole and that thioformamide caused a marked increase in SUN concentration when administered to mice in combination with BSO.

N-alkylformamides are metabolized to N-alkylcarbamoylating species by hepatic microsomes from rodents and humans.

Hepatotoxic formamides such as N-methylformamide (NMF) and N,N-dimethylformamide (DMF) are metabolized in vivo to N-acetyl-S-(N-methylcarbamoyl)cysteine via oxidation at the formyl carbon, which yields a reactive intermediate. The hypothesis was tested that this biotransformation route can be studied in vitro with hepatic fractions. NMF was incubated with microsomes or cytosol obtained from BALB/c mice, and metabolically generated N-methyl-carbamoylating species were analyzed after derivatization with ethanol in base to furnish ethyl N-methylcarbamate. Generation of metabolite was catalyzed by microsomes, but not by cytosol. Detection of the N-methylcarbamoylating species was dependent on the presence in the incubation mixture of NMF, viable microsomes, NADPH, and a thiol-containing agent such as glutathione. Metabolite formation was inhibited by SKF 525-A (3 mM) and abolished when the incubation atmosphere consisted of an air/carbon monoxide mixture (1:1) instead of air. Metabolism was not induced by pretreatment of mice with phenobarbital or beta-naphthoflavone. N-Ethylformamide and the DMF metabolite N-(hydroxymethyl)-N-methylformamide, but not DMF, were metabolized by microsomes to the N-alkylcarbamoylating metabolite at a measurable rate. NMF metabolism was also observed with liver microsomes from Sprague-Dawley rats or from humans. In the case of rat microsomes the rate of metabolism was half of that measured with murine microsomes. The results suggest that (i) the metabolic toxification of NMF can be studied in hepatic microsomes and (ii) the oxidation of the formyl moiety in N-alkylformamides is catalyzed by cytochrome P-450.

2-week inhalation study of N-monomethylformamide in rats.

N-Monomethylformamide (MMF) is a chemical intermediate with potential for inhalation exposure in humans. Human exposures to MMF have occurred in cancer chemotherapy but have been limited due to liver damage. To assess the toxicity of MMF, groups of 15 male rats each were exposed by nose-only inhalation, 6 hr/day, 5 days/week, for 2 weeks to either 0 (control), 50, 130, or 400 ppm MMF. Five rats per group were killed following the 10th exposure, five were killed after a 14-day postexposure recovery period, and five rats were used to determine urinary MMF excretion. Parameters investigated were clinical observations and body weights, clinical pathology, and gross and microscopic pathology including organ weights. Liver damage occurred in rats exposed to either 130 or 400 ppm. This was detected both by increases in serum enzyme activity indicative of liver injury and by microscopic changes in the liver. The changes were more severe in the 400-ppm rats and were partially reversible. Other organs were not adversely affected by inhalation of MMF. The amount of MMF excreted in the urine was dependent on the exposure concentration and MMF was present 14 days postexposure at the higher exposure levels. The no-observed-effect level under the conditions of this experiment was 50 ppm.