Search

Sodium picramate is the sodium salt of picramic acid, a substituted phenolic compound. Sodium picramate and picramic acid function as hair colorants; they are reportedly used in 31 and 3 hair-dye products, respectively. No concentration-of-use data were available for sodium picramate, but picramic acid was reported to be used at 0.6%. The Cosmetic Ingredient Review Expert Panel recognized that adding picramic acid to a hair-dye formulation likely results in formation of a salt such as sodium picramate, which suggested that safety test data for one ingredient would be applicable to the other. Hair dyes containing these ingredients bear a caution statement and patch test instructions for determining whether the product causes skin irritation. The panel finds that the available data support the safety of these colorants in hair dyes and expects that sodium picramate would be used at concentrations comparable to those reported for picramic acid.

Piper methysticum leaf/root/stem extract is the cosmetic ingredient name for a material derived from the leaves, roots, and stems of the Piper methysticum G. Forster plant, commonly known as kava kava. This and other kava-derived ingredients are used as skin-conditioning agents at concentrations from 0.0001% to 0.1%. The Food and Drug Administration issued a consumer advisory in 2002 expressing concern about liver damage in individuals who have ingested kava products. The available oral toxicity data support the concern about liver damage on ingestion but do not resolve the question, for example, whether these ingredients would be substantially absorbed through the skin. Other data needs are described, including toxicology data for yangonin, methysticin, and kavain, which may be present in kava-derived ingredients. Accordingly, the available data are insufficient to support the safety of these ingredients in cosmetics.

PPG-2 methyl ether, PPG-3 methyl ether, and PPG-2 methyl ether acetate are used in cosmetics as fragrance ingredients and/or solvents at concentrations of 0.4% to 2%. Propylene glycol ethers are rapidly absorbed and distributed throughout the body when introduced by inhalation or oral exposure, but the inhalation toxicity of PPG-2 methyl ether vapor, for example, is low. Aerosols, such as found with hair sprays, produce particle sizes that are not respirable. Because these ingredients are highly water-soluble, they are likely to be absorbed through the human skin only at slow rates, resulting in low blood concentrations and rapid removal by the kidney. These ingredients are not genotoxic and are not reproductive or developmental toxicants. Overall the data are sufficient to conclude that PPG-2 methyl ether, PPG-3 methyl ether, and PPG-2 methyl ether acetate are safe as used in cosmetics.

A synthetic DNA fragment was constructed to determine the effect of 5' and 3' neighbors of guanine runs on the binding of chemical carcinogens. Determinations were made on the relative intensity of reactivity between aflatoxin B1 or benzo(a)pyrene and methylnitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with various guanine positions in an endlabeled DNA fragment of known sequence. After reaction, the fragments were depurinated to produce strand breaks to allow Maxam and Gilbert sequencing for guanine positions. Relative reaction intensities were compared densitometrically. 3' neighbors exerted greater influence on carcinogen binding than did 5' neighbors, the influence extended only to the adjacent guanine and depended upon the chemical nature of the carcinogen. In addition, the presence of one carcinogen adduct in the guanine run influenced the formation of a subsequent adduct when the DNA was exposed to a second carcinogen, and this effect also depended on the nature of the second carcinogen. The results suggest that DNA adduct formation in the presence of multiple carcinogens is more complicated than an additive mechanism would suggest.

The most frequent mutation in human hepatocellular carcinoma (HCC) in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is a mutation in codon 249 of the p53 gene. Schistosomiasis is known to cause p53 mutation. We hypothesized that the combination of schistosomiasis and aflatoxin B1 increases the incidence of p53 gene mutation.Liver tissue from 21 patients with schistosomiasis and 5 patients without schistosomiasis were analyzed for occurrence of mutations of the p53 gene and levels of N7-guanine-AFB1 adducts.The presence of mutations in codon 249 of p53 gene was higher in patients infected with Schistosoma haematobium (S. haematobium) than in those infected with Schistosoma mansoni (S. mansoni) or a combination of both strains (p0.01), compared to control subjects. No mutations were detected in p53 gene in liver DNA from schistosomiasis-free patients. Significant amounts of N7-guanine-AFB1 adducts and novel adenine-adducts (p0.01) were detected in patients with schistosomiasis, mostly in patients infected with S. haematobium or a combination of both strains, compared to control subjects.These data suggest that schistosomiasis and exposure to aflatoxin B1 act synergistically to increase the incidence of p53 gene mutation. The increase in p53 mutations may enhance progression of HCC at an early age in patients with schistosomiasis.

Hydrazine sulfate is a genotoxic hepatocarcinogen for the hamster. A study was conducted to follow changes in DNA maintenance methylation in selected genes in liver DNA during the 21-month induction of liver adenomas and hepatocellular carcinomas by demonstrating changes in restriction fragment length polymorphism. Male Syrian golden hamsters were exposed to hydrazine sulfate in the drinking water at three concentrations (170, 340 and 510 mg/l) shown previously to result in a dose-dependent induction of liver tumors. Liver DNA from animals exposed to the high concentration for 6, 12, 16, 20 and 21 months and animals exposed to the low or mid concentration for 21 months was digested with EcoRI, MspI, HindIII or BamHI, or a combination of one of these endonucleases and a methyl-sensitive restriction enzyme, HpaII or HhaI. The DNA digests were subjected to Southern analysis using a c-DNA probe for one of the following genes: DNA methyltransferase (DMT), c-Ha-ras, c-jun, c-fos, and c-myc proto-oncogenes, p53 tumor suppressor gene or gamma-glutamyltranspeptidase. Alteration in DNA restriction by methyl-sensitive endonucleases was detected in four (DMT, c-Ha-ras, p53 and c-jun) of the seven genes examined and as early as 6 months in animals exposed to the highest concentration of hydrazine sulfate; alteration of recognition sites in c-Ha-ras was also detected in DNA from animals exposed for 21 months to the intermediate concentration of hydrazine sulfate. Early changes in recognition sites, presumed to indicate altered methylation status of DNA cytosine and/or guanine mutations, were seen using c-DNA probes for DMT, c-Ha-ras and c-jun; in the p53 tumor suppressor gene alteration of such sites was a late event relevant to appearance of liver adenomas and hepatocellular carcinomas. Evidence for hypomethylation in the p53 and c-jun genes and hypermethylation of the c-Ha-ras and DMT genes is provided. This study supports the induction of site-specific hypomethylation and hypermethylation during the course of hydrazine carcinogenesis.

Hydrazine, which is toxic and carcinogenic to rodent liver, has been shown to react with endogenous formaldehyde in the liver to form formaldehyde hydrazone (CH 2 = N-NH 2 ), an alkylating intermediate that methylates DNA guanine at the N7- and O 6 -positions. Studies were conducted to investigate the role of chronic hydrazine-induced hepatotoxicity on DNA maintenance methylation (formation of 5-methyldeoxycytosine) and the development of liver cancer. Male Syrian golden hamsters were given hydrazine sulfate (0, 170, 340 and 510 mg/l) in drinking water for 21 months (average dose 0, 4.2, 6.7 and 9.8 mg/kg body wt hydrazine as the free base). Hepatotoxicity was evaluated histologically, and regenerative DNA synthesis and maintenance methylation were measured as the incorporation of [methyl- 14 C]thymidine into DNA and the methyl moiety of [methyl- 3 H]methionine into 5-methyldeoxycytosine in DNA, respectively. Methylguanines were detected in liver DNA at the first observation time of 6 months of treatment ; levels of these aberrant bases decreased or became undetectable at 14 months, and increased in a dose-related manner for the remainder of the study. DNA adducts persisted in the highest dose group throughout the study, repeating the results of a similar study previously reported by this laboratory (Bosan et al., Carcinogenesis, 8, 439-444, 1987). Linear regression analysis of thymidine and methionine methyl moiety incorporation into liver DNA suggested impairment of maintenance methylation of DNA (5-methyl-deoxycytosine) in the middle and high exposure animals. Hepatic adenomas and hepatocellular carcinomas developed in a dose-related manner and were highly correlated to decreased uptake of radiolabel from methionine into DNA 5-methylcytosine. These results are part of a continuing study on alteration of maintenance methylation during hydrazine induction of liver cancer.

The effect of a carcinogen-DNA adduct on the formation of a second adduct upon subsequent exposure to a second carcinogen was studied using (i) a modified Maxam-Gilbert chemical sequencing reaction and (ii) a DNA synthesis termination analysis. A DNA fragment of known sequence was reacted with micromolar concentrations of N-methyl-N-nitrosourea (MNU), (+)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF), or aflatoxin B 1 -8,9-epoxide (AFB 1 epoxide) singly or in successive double reactions. N-Acetoxy-AAF adducts were sensitive to the Maxam-Gilbert sequencing reaction for guanine ; these adducts significantly blocked the formation of BPDE-guanine adducts. Treatment of DNA with BPDE, however, did not inhibit subsequent formation of N-acetoxy-AAF adducts. DNA synthesis termination analysis suggested that methylation of guanine altered subsequent arylation of guanine by N-acetoxy-AAF and AFB 1 epoxide, and that combined treatment inhibited replication, an effect not seen after MNU treatment alone. Possible mechanisms for the modulation of carcinogen binding are discussed. It is concluded that not only is DNA damage by genotoxic carcinogens dependent upon both the chemical nature of the carcinogens and the nearest neighbors to target guanine bases, but that the effect of subsequent exposures to carcinogens is not always additive.

Effects on N-methyl-N-nitrosourea (MNU) mediated methylation of the N7 position of guanine were compared in defined sequences of DNA containing cytosine or 5-methylcytosine (5mC) using a Maxam-Gilbert sequencing technique. Cytosine methylation in 5'-CpG-3' pairs within a subcloned fragment of the 5' region of the human HPRT gene was generated with SssI methylase and S-adenosylmethionine. Cytosine methylation was demonstrated by both the inhibition of DNA restriction by methylation sensitive endonucleases and the lack of cleavage at 5-methylcytosines by hydrazine. MNU-dependent methylation of the N7 position of guanine was inhibited up to 18% when 5mC was a 5' neighboring base to guanine and was inhibited up to 36% in an alternating CpG region in which both 5' and 3' neighboring bases of guanine were enzymatically altered to 5mC. It can be concluded that 5-methylcytosine has discernible effects on MNU methylation of the N7 position of specific guanine bases in DNA.

The rate of formation and the persistence of an exocyclic guanine adduct formed in DNA of rodents treated with various doses of N-nitrosopyrrolidine (NPYR) have been determined. NPYR is hepatocarcinogenic to the rat and forms a covalent adduct in liver DNA; this adduct was recently identified as 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2, 1-f]purine-4[3H]-one. Dose-dependent amounts of adduct formed in liver, kidney and lung DNA of rats, hamsters and mice given oral doses (56-900 mg/kg body wt) of NPYR. The persistence of the adduct in DNA after administration of low doses of NPYR to rats was greatest in the target organ, i.e. the liver; at high doses of NPYR, adduct levels in DNA changed little over a period of at least 72 h. In the hamster, in which NPYR is carcinogenic to the lung but apparently not the liver, the adduct level in liver DNA was an order of magnitude greater than in lung or kidney DNA for a dose of NPYR of 225 or 900 mg/kg body wt; persistence of the adduct in lung DNA was only slightly longer than in liver DNA. The formation and persistence of the 7,8-pyridoguanine adduct in the rat appeared to be consistent with the organotropy of this carcinogen, but this was not true for the hamster, a species that seems to be more resistant to induction of liver and kidney cancer by this carcinogen. Imidazole, an inhibitor of microsomal amine oxidase, and disulfiram, an inhibitor of aldehyde dehydrogenase, decreased metabolic activation of NPYR to an alkylating intermediate; inducers and inhibitors of cytochrome P450 monooxygenases had little effect on the metabolic activation of NPYR to an alkylating agent.

Hydrazine is acutely neurotoxic, hepatotoxic and nephrotoxic; it is also carcinogenic to liver and lung in rodents. Administration of hydrazine results in formation of 7-methylguanine and O6-methylguanine in target organ DNA of rats, mice, hamsters and guinea-pigs. It has been suggested that hydrazine reacts with endogenous formaldehyde to form a condensation product which could be metabolized to a methylating agent. Solutions of 0.50 mM hydrazine and formaldehyde have, upon mixing, NMR spectra (300 MHz) consistent with the formation of formaldehyde hydrazone but not other possible condensation products such as tetraformyltriazine or formaldehyde azine. These same solutions evidencing hydrazone formation, when incubated in an in vitro system containing post-mitochondrial (S9), microsomal, cytosolic or mitochondrial cell fractions, resulted in the methylation of DNA guanine; S9 was the most active fraction. Neither the P-450 monooxygenase nor flavin monooxygenase systems appeared to be important in hydrazine/formaldehyde-induced methylation of DNA. However, sodium azide, cyanamide and carbon monoxide all inhibited S9-supported DNA methylation. Bovine liver catalase, a heme-containing cytochrome, readily transformed hydrazine/formaldehyde to a methylating agent. The data support formation of formaldehyde hydrazone as the condensation product of hydrazine and formaldehyde which is rapidly transformed in various liver cell fractions, perhaps by catalase and/or catalase-like enzymes, to a methylating agent.

The formation of N-nitrosopyrrolidine from sodium nitrite and pyrrolidine in vivo in a dog's stomach is demonstrated. The rate of formation of nitrosopyrrolidine is shown to be subject to pronounced catalytic effects. Nitrosopyrrolidine is also shown to disappear rapidly from the stomach, probably due to absorption.

The formation and persistence of O6-methylguanine and 7-methylguanine in colon, kidney, and liver DNA were measured over a period of 25 weeks during 1,2-dimethylhydrazine (SDMH) carcinogenesis. Rats were given 14 weekly s.c. injections of 21 mg SDMH/kg body wt., and methylated guanines in DNA were determined quantitatively one week after each injection to measure the long-term accumulation of these aberrant bases. No accumulation of either base was seen in colon DNA, and no O6-methylguanine was seen to accumulate in liver DNA. The concentrations of O6-methylguanine and 7-methylguanine in kidney DNA did increase with reported administration of the carcinogen, but these bases were removed within six weeks after the last (14th) injection of SDMH. Large amounts of 7-methylguanine accumulated in liver DNA over the 14-week treatment period. The concentration of 7-methylguanine in liver and kidney DNA increased at rates greater than could be accounted for by using kinetic relationships determined in single-exposure studies. The concentration of O6-methylguanine in kidney DNA also increase at a rate greater than would be expected from calculations based on the rate of removal following a single administration of SDMH.

Cytochalasin E, a minor toxic metabolite of the fungus Aspergillus clavatus, is acutely toxic to rats, mice, and guineapigs. The LD50 values for a single ip administration of cytochalasin E were: 1-day-old rats, 0.98 mg/kg; adolescent rats, 2.60 mg/kg; mice, 4.60 mg/kg, and guinea-pigs, 0.5–1.5 mg/kg. The toxicity of cytochalasin E was reduced in adolescent rats when administered orally (LD50 9.10 mg/kg) and was increased when administered by the intrathoracic route (LD50 1.30 mg/kg). Rats receiving a fatal ip dose of cytochalasin E died within 2–18 hr with 2–3 ml fluid in the peritoneal cavity. Intrathoracic administration of cytochalasin E killed rats within 2–8 hr and resulted in accumulation of 2.5–3.5 ml pleural fluid. Rats receiving the toxin orally died within 4–18 hr with 1.0–1.5 ml gastric fluid. Histopathologic examination revealed congestive degenerative changes, necrosis of liver, kidney, spleen, pancreas, and small intestine, brain edema, pulmonary hemorrhages, and injury to vascular walls.

Liver DNA of young adult male Fischer 344 rats given orally a necrogenic dose of hydrazine (60 mg/kg body wt) rapidly became methylated at the 7-position of guanine, and more slowly and to a lesser extent at the O 6 -position of guanine. The identity of 7-methylguanine from liver DNA of hydrazine-treated rats was confirmed by four high-pressure liquid chromatographic methods, in one paper chromatography system, and by ultraviolet absorption spectrometry. Similar evidence was obtained to support the identification of O 6 -methylguanine. Liver DNA isolated from rats given hydrazine and [ methyl - 3 H]methionine contained 7-methylguanine and O 6 -methylguanine which were labeled to a greater extent than was the parent base, guanine, suggesting that S -adenosylmethionine was the methyl source in the alkylation of DNA. Aberrant methylation of liver DNA was also observed in rats after administration of hepatotoxic doses of carbon tetrachloride and ethanol, indicating that the DNA methylation may be a nonspecific response to chemical injury to the liver.

A single lethal dose of cytochalasin E (3.50 mg/kg ip) produced a rapid decrease in plasma protein concentrations of adult male Fischer rats (250–300 g). Plasma concentrations of albumin and globulin were reduced within 20 min without significant change in plasma fibrinogen concentration. Reduction of plasma volume paralleled the appearance of fluid at the site of injection; these changes were dose-dependent. At a dose of 1.0 mg/kg (ip) loss of plasma protein occurred in 3 hr; the protein content gradually returned to control values in the following 20 hr. At the same time, there was an accumulation of fluid in the pleural cavity which reached a maximum when the plasma volume had returned to the control value. The intraperitoneal administration of cytochalasin E produced a dose-dependent decrease in mean arterial pressure. There was a biphasic response in plasma glucose concentration following a lethal dose (3.50 mg/kg, ip) of cytochalasin E, a hyperglycemia during the first hour after toxin treatment, followed by a mild hypoglycemia. Liver glycogen content was reduced to 9% of control values within 60 min after treatment. Cytochalasin E appears to act directly on the walls of blood capillaries permitting extravascular effusion of plasma fluid, albumin, and globulin; cause of death is apparently due to shock.

Aflatoxins are associated with acute toxicoses in poultry and livestock and with liver cancer in human populations in Africa and Southeast Asia. Comparative metabolism studies indicate that aflatoxin B1, the most potent of these compounds, requires metabolic activation to the ultimate carcinogen. The mycotoxin (1) is oxidatively demethylated to form the phenolic derivative, (2) is hydroxylated directly at three sites, and (3) undergoes reduction of the carbonyl group in the cyclopentenone ring to a hydroxyl group; these five hydroxy derivatives all appear to be part of detoxication pathways. Considerable evidence is now available to support the hypothesis that activation of aflatoxin B1 to the ultimate carcinogen involves epoxidation of the double bond in the terminal furan ring. The epoxide decomposes to form a carbonium ion at C-2, which attacks nucleophilic sites in DNA, especially on the guanine moiety. Thus, like carcinogenic polycyclic hydrocarbons and N-nitroso compounds, aflatoxin B1 appears to be activated to an alkylating agent.

Kinetics of ethylation of target and non-target organ DNA in vivo by diethylnitrosamine (DEN) was compared in rats and Syrian golden hamsters, since published reports indicate a single dose of DEN induces both kidney and liver tumors in rats and almost exclusively respiratory tract tumors in hamsters. Following treatment with 200 mg DEN/kg, 7-ethylguanine (7-etG) was lost more rapidly from hamster than from rat liver DNA, while O6-ethylguanine (O6-etG) persisted longer in hamster than in rat liver DNA. DNA ethylation was not detected in rat lung (non-target organ), while both 7-etG and O6-etG were quantitated in hamster lung (target organ) following DEN treatment. DNA ethylation in rat kidney DNA was approximately 1/10 of that in liver by 200 mg DEN/kg, and the persistence of 7-etG and O6-etG differed only slightly in these tissues. Ethylation of hamster liver DNA by DEN at doses between 20 and 200 mg/kg, as measured by 7-etG and O6-etG was proportional to the dose of carcinogen up to 160 mg/kg; at larger doses DNA ethylation sharply increased. Differences in the persistence of O6-etG between DEN-treated rats and hamsters cannot solely account for species differences in the organotropism of DEN carcinogenesis.

Administration of the hepatotoxin and carcinogen, inorganic hydrazine, to rodents results in the formation of 7-methylguanine and O6-methylguanine in liver DNA; co-administration of [methyl-14C]methionine or [14C]formate with the hydrazine labels the methylguanines, suggesting involvement of the 1-carbon pool in the methylation process. The present study investigates the proposal that the methylation mechanism involves reaction of hydrazine with endogenous formaldehyde to yield formaldehyde hydrazone, which could be metabolized to the potent methylating agent diazomethane. Hamsters were pretreated with methanol, ethanol or cyanamide to alter the endogenous hepatic aldehyde levels prior to administration of hydrazine. Formaldehyde levels were refractory to the pretreatments; hepatic acetaldehyde levels were increased, but hydrazine administration under such conditions did not result in the formation of ethylated guanines in DNA. Methanol and ethanol inhibited hydrazine-induced methylation of DNA. Hydrazine incubated with liver S9 fraction and calf thymus DNA induced the formation of 7-methylguanine and O6-methylguanine when formaldehyde was present in the incubation system; substitution of formaldehyde with acetaldehyde in the incubation medium did not result in any detectable alkylation of DNA. Both liver microsomal and cytosolic fractions demonstrated heat-labile activity in supporting the hydrazine-induced methylation process. Tetraformyltrisazine, or a similar reaction product of hydrazine and formaldehyde, may be a more important intermediate than formaldehyde hydrazone in the hydrazine-induced methylation of DNA.

Liver DNA isolated from rats given N-nitrosopyrrolidine contained amounts of an unidentified fluorescent adduct which were dose dependent. The adduct formed gradually over the first 12 hr after administration and was slowly removed from the DNA in vivo . Fluorescence and chromatographic properties of the adduct suggested the compound was a substituted guanine; also, the putative adduct was readily removed from DNA by neutral thermal hydrolysis, as are 3-alkyladenines and 7-alkylguanines. Evidence was also obtained for the formation of 7-methylguanine in liver DNA of N-nitrosopyrrolidine-treated rats; however, N-nitrosopyrrolidine was not the methyl source for this alkylated guanine.

(1974). Isolation and Purification of Cytochalasin E and Two Tremorgens from Aspergillus Clavatus. Plant Foods for Man: Vol. 1, No. 2, pp. 113-119.

Fischer 344 or Sprague Dawley rats were fasted overnight and given orally 30--90 mg hydrazine/kg body wt. The presence of 7-methylguanine and O6-methylguanine in liver DNA was demonstrated 5 and 24 h after hydrazine administration using two different analytical techniques. Methylation levels changed little with dose except for the highest dose (ca. LD50) at which the levels doubled. In a time-response study, rats were given 90 mg hydrazine/kg body wt. and killed 0.25 to 96 h later. Both 7-methylguanine and O6-methylguanine were detected quantitatively in liver DNA from rats as early as 15 min after hydrazine administration. After maximum levels of methylguanines had formed, 7-methylguanine was removed from DNA at a rate of approximately 50% in 47 h; the half-life of O6-methylguanine in liver DNA was approximately 13 hr. Three or four, but not one or two, daily administrations of 3 mg hydrazine/kg body wt. also produced detectable levels of 7-methylguanine in rat liver DNA. Neither 7-methylguanine or O6-methylguanine was detected in comparable amounts of liver DNA from control animals. The study confirms the observation that hydrazine administration results in the formation of methylated guanines in liver DNA.

The acute and chronic toxicity of N -nitrosamines and N -nitrosamides are reviewed in general terms. Emphasis is made of the relationship between metabolism and toxicity.

Hydrazine is carcinogenic to the mouse and rat, but three earlier studies have reported no carcinogenicity of hydrazine in the hamster. Administration of hydrazine to mice, rats and hamsters results in rapid methylation of liver DNA guanine for which endogenous formaldehyde appears to be the source of the methyl moiety. Hamsters were given hydrazine sulfate at 170, 340 and 510 mg/l in the drinking water for 2 years [average dose of 4.6, 8.3 and 10.3 mg hydrazine (free base)/kg body wt over the 2-year period], during which levels of methylation of DNA guanine in liver, kidney and lung, and histopathologic examinations of these tissues were carried out; dimethylnitrosamine, as a positive control, was administered at 10 mg/l in the drinking water (average dose of 1.1 mg/kg body wt over the 4-month measurement period). Both 7-methylguanine and O6-methylguanine were readily detectable at 6 months exposure in hamsters given hydrazine or dimethylnitrosamine; in hydrazine-treated animals only trace amounts of these bases could be detected after 12 months exposure; these bases were again detected in liver DNA at exposure times of 18 and 24 months. Hepatocellular carcinomas were observed in hamsters treated at the highest dose of hydrazine sulfate after 78 weeks of exposure; the incidence of liver cancer was dose-related over the course of the experiment: 32% for hamsters exposed to 510 mg hydrazine sulfate/l, 12% for 340 mg/l and none at 170 mg/l. Hamsters given dimethylnitrosamine developed high levels of 7-methylguanine and even higher levels of O6-methylguanine and both liver cholangiocellular carcinomas (73% incidence), as reported before, and hepatocellular carcinomas (27% incidence), a new finding. These results demonstrate for the first time that hydrazine is a liver carcinogen in the hamster and provide new information regarding the accumulation of DNA damage during the entire induction period for the carcinomas.

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O 6 -methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.

As the initial phase of a survey to evaluate the relationship between dietary loads of aflatoxin and the incidence of liver cancer in South-east Asia, this study demonstrated the distribution of moulds in foods of Thailand and Hong Kong and distinguished those capable of mycotoxin production. Mycological studies of more than 3000 food samples showed Aspergillus to be the commonest contaminating fungus, with Aspergillus flavus the predominating species. Other fungi included species of Penicillium, Fusarium and Rhizopus. Bioassays evaluating acute and subacute toxicity to rats identified the infecting agents capable of producing mycotoxins other than aflatoxins. Among 162 isolates, 49 proved to be toxigenic, and these included species of all the genera commonly identified in food samples.

Twenty-four young female macaque monkeys were given 0, 0.5, 1.5, 4.5, 13.5, or 40.5 mg of crystalline aflatoxin B 1 per kg body wt by oral administration. The LD50 for aflatoxin B 1 was 7.8 mg/kg for a test period of 148 hr. Aflatoxin B 1 could be detected in brain, liver, kidney, heart, blood, and bile in some of the animals 148 hr after toxin administration. The response of these monkeys to acutely toxic doses of aflatoxin B 1 was strikingly similar to an acute children's disease of unknown etiology seen in Thailand.

The incidence of primary liver cancer was determined in two selected populations in Thailand by direct field investigation. Liver viscerotomy specimens were obtained from 21% of the persons aged 15 yr or more who lived and died in the municipal areas of Ratburi, Potharam, Baan Pong and Nakorn Pathom (combined population 99,537) in south-western Thailand, and from 33% of comparable persons in the municipal areas of Songkhla and Haad Yai (combined population 97,867) in southernmost Thailand. Incidence, as measured in this survey, was six new cases/100,000 people/yr in the first (south-west) area and two new cases/100,000 people/yr in the second (Songkhla-Haad Yai) area.

Duration and point prevalence of primary liver cancer in rural regions of Thailand were determined to provide a basis for calculating the incidence of this condition. Hospital records of 271 established cases of primary liver cancer in Thailand were reviewed to estimate the usual duration of the disease among Thai patients. Duration is defined here as the interval between first recognition of the disease by a physician and date of death. Mean duration for 184 histologically proven and clinically confirmed cases was 1·3 months; for all cases, including an additional 87 identified only by clinical diagnosis, the interval was 1·1 months. Associated cirrhosis did not affect duration. In two rural regions of Thailand, examination demonstrated a high prevalence of hepatomegaly among 53,076 persons aged 15 yr or more, the age range for primary liver cancer. No case of primary liver cancer was found, a result in accord with the known incidence of the disease. In Singburi, reported to be an area of high liver-cancer incidence and high aflatoxin ingestion, the hepatomegaly rate was 2·7/1000 in persons aged 15 yr or more. The rate in males was twice that in females. In Songkhla, an area where both cancer incidence and aflatoxin ingestion were low and where the overall hepatomegaly rate was 3·4/1000, the male:female ratio was the reverse of that in Singburi. Primary cancer of the liver in Thailand predominates in males in a proportion of 4 to 1.

1. The effects of various doses of dimethylnitrosamine on the hydrocortisone induction of tryptophan pyrrolase were studied. A single LD50 dose of dimethylnitrosamine inhibits the synthesis of the enzyme if given within the first 5hr. of the induction. A quarter of this dose also inhibits the synthesis of the enzyme, but, in addition, retards the rate of decay of the enzyme. 2. A single small dose of dimethylnitrosamine significantly inhibits the hydrocortisone induction of tryptophan pyrrolase for at least 14 days without causing widespread damage to liver substructure. 3. The inhibition by dimethylnitrosamine of induced tryptophan pyrrolase synthesis is probably independent of any action of the toxin on the synthesis of cofactors necessary for full expression of the enzyme's activity. Dimethylnitrosamine appears to act directly on the synthesis of the enzyme protein. 4. The synthesis of that form of the enzyme which is most sensitive to hydrocortisone is apparently also most susceptible to the action of dimethylnitrosamine. 5. It is suggested that the inhibition of protein synthesis by dimethylnitrosamine may be a result of methylation of messenger RNA, which then is unable to code effectively for amino acid polymerization.

Aflatoxin consumption through cooked foods was determined by three 2-day surveys over a period of 1 yr for 144 randomly selected households in nine villages within three areas of Thailand. The Singburi and Ratburi areas had the highest levels of aflatoxin ingestion, with respective annual means ranging from 73 to 81 and from 45 to 77 ng total aflatoxins/kg body weight/day on a family basis. Intakes as high as 1072 ng total aflatoxins/kg body weight/day were recorded for individuals. Average annual aflatoxin consumption in Songkhla was 10–14 times less than in Singburi. The Singburi dietary load of aflatoxin represents 20–30% of intake values that induce a liver-tumour incidence of nearly 100% in rats following continuous exposure.

Long-term feeding studies were conducted in Sprague-Dawley rats using various dietary concentrations (up to 1000 ppm each) of nitrite and morpholine or 5 or 50 ppm N -nitrosomorpholine. The animals fed nitrite and morpholine at moderate to high concentrations developed hepatocellular carcinomas and angiosarcomas, histologically identical to those induced by preformed N -nitrosomorpholine. The data suggest that in vivo nitrosation of morpholine does occur in the rat, presumably in the acidic gastric environment.

In the weanling male rat, a single oral LD20 of aflatoxin B1 caused a transient inhibition of leucine incorporation into liver proteins. The brief (6-hour) suppression was followed by a prolonged period of stimulation of hepatic protein synthesis which persisted for 3–6 days after dosing. Liver deoxyribonucleic acid metabolism was not altered during this period. Five consecutive daily administrations of sublethal doses of the toxin, each equivalent to 10% of the LD50, caused significant suppression of growth and liver weight in the duckling. Liver glycogen content and glycogenesis were also decreased, whereas lipid content was elevated. Treatment of weanling male rats according to the same dosing schedule resulted in suppression of growth and lowering of food intake, efficiency of food utilization, and liver weight; but liver composition was not significantly altered in this species.

Hydrazine (H2N-NH2) is a strong reducing agent, widely used in large amounts as an anticorrosive agent, as an intermediate in chemical syntheses, and as a propellant fuel. The compound at a single, high dose is neurotoxic, producing convulsions and respiratory arrest; at a lower single dose, hydrazine produces marked damage to the liver and kidneys (1,2). In a number of in vitro and in vivo tests, hydrazine, at relatively low concentration (dose), was positive as a mutagen (3–7). Chronic oral administration of hydrazine, at doses which depressed growth rate, produced pulmonary carcinomas in mice (8) and hepatocellular carcinomas and lung adenocarcinomas in rats and mice (9). When administered by inhalation at four different doses to rats, mice, and hamsters for 6 hr/d, 5 d/wk for 1 yr, hydrazine (only at the maximum tolerated dose) induced microscopic squamous cell carcinomas in the nasal turbinates of rats but no tumors in mice or hamsters (10). Hydrazine is capable of reacting in vitro under severe conditions with pyrimidine bases in DNA nucleotides to form the dihydro- or 4-hydrazino-derivatives or to open the pyrimidine rings (11). No one has yet been able to demonstrate a hydrazine-DNA adduct under in vivo conditions.

Earlier studies demonstrated that hydrazine administration to rodents results in the methylation of liver DNA guanine and that S-adenosylmethionine was the apparent methyl donor in this response. The present study characterized the hamster as an animal model in a study of the methylation response to acute and subchronic exposures to hydrozine and has examined further the possible roles S-adenosylmethionine and the 1-carbon pool may have in the methylation of liver DNA guanine in hydraine-poisoned animals. The hamster is more resistant that rate or mouse to the acute toxicity of hydrasine and can tolerate up to 120 mg hydrazine/kg body wt per os. The amounts of 7-methylguanine that formed in liver DNA changed little when the dose of hydrazine ( 60 mg/kg) the formation of these aberrant basses in liver DNA increased greatly, as did the cytotoxicity in the liver. The rate of formation of these bases after administration of 90 mg hydrazine/kg body wt was rapid and 06-methylguanine persisted in hamster liver DNA longer than it did in rat liver DNA, suggesting that repeated doses of hydrazine to the hamster may force an accumulation of the promutagenic lesion in the DNA.

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.

Long-term feeding studies were conducted in Sprague-Dawley rats and Syrian golden hamsters using various dietary concentrations of nitrite and morpholine (up to 1000 ppm of each) or N -nitrosomorpholine (5 or 50 ppm). Most combinations of the two chemicals induced a high incidence of hepatocellular carcinoma in rats and a lower incidence in hamsters. The highest level of nitrite and morpholine (1000 ppm of each) had a stronger potential for carcinogenesis in both rats and hamsters than did a dietary level of 50 ppm of preformed N -nitrosomorpholine. Nitrite and morpholine also induced angiosarcomas in both species, most frequently in the liver, with the lung as the next most common site. The nitrite concentration in the diet seemed to have a greater effect on the incidence of hepatocellular carcinoma and angiosarcoma in the rat than did the concentration of morpholine. Dietary concentrations of 5 ppm each of nitrite and morpholine induced hepatocellular carcinoma and angiosarcoma in some rats. High concentrations of sodium nitrite alone were associated with a relatively high incidence of lymphoreticular tumours.

Hydrazine, H2N-NH2, is widely used in industry and the military; it is acutely toxic to the liver, kidney, and central nervous system. Chronic inhalation of hydrazine produced squamous cell carcinomas of the nasal turbinates in Fischer 344 rats but only at exposures which led to severe rhinitis with focal hyperplasia and squamous metaplasia early in the experiment; Syrian golden hamsters and C57B16 mice did not develop cancers under the same conditions. Biancifiori and Severi (1966) showed that oral administration of hydrazine at growth-depressing doses induced hepatocellular carcinomas and lung adenomas in male rats, pulmonary adenomas in BALB/c/Cb/Se and BALB/c/An/Se mice and pulmonary adenocarcinomas and hepatomas in CBA/Cb/Se mice. Swiss Webster (Crl:CFW(SW)BR) and B6C3Fl(:CrlBR) male mice from Charles River Breeding Lab. in Massachusetts, USA, were fasted overnight and given 0, 5,10,20, or 40 mg hydrazine per kg body weight by intraperitoneal injection and decapitated 24 hr later. Liver DNA was isolated and analyzed by high performance liquid chromatography and fluorescence spectrophotometry for chemical adducts involving the purine bases. A dose related formation of 7-methylguanine and O6-methylguanine was observed in DNA, with little difference between the two strains of mice. Evidence suggests that the hydrazine reacts with endogenous formaldehyde to form a stable intermediate, possibly tetraformyltrisazine, which is metabolized by alcohol and aldehyde dehydrogenases to an active methylating agent. In an experiment similar to the dose-response study, 20 mg hydrazine/kg body wt was given to the two strains of mice, and animals were killed at 24 hr intervals to determine the persistence of these aberrant purines in DNA. The rates of formation of 7-methylguanine and O6-methylguanine and the rates of disappearance of 7-methylguanine from DNA were the same in both strains. The rate of disappearance of the promutagenic base, O6-methylguanine, however, was an order of magnitude slower in the B6C3F1 mouse compared with the Swiss Webster mouse; the half-life of O6-methylguanine in the B6C3F1 mouse liver DNA was greater than 200 hr, while in Swiss Webster mouse liver DNA, it was 17 hr. These results are compared with a similar study in the Syrian golden hamster in which the levels of O6-methylguanine in liver DNA were measured over a 2-yr exposure to hydrazine; the results suggest that the B6C3F1 mouse would be highly susceptible to the hepatocarcinogenicity of hydrazine. This susceptibility is compared with that of other species.

Formation of 7-methylguanine and O6-methylguanine in liver DNA was measured 24 hr after administration of various single oral doses of 15 to 120 mg hydrazine/kg body wt to Syrian golden hamsters, and at various times up to 96 hr after a single oral dose of 90 mg hydrazine/kg body wt. Formation of these bases in hamster liver DNA appeared similar to that reported earlier for rats and mice treated with necrogenic doses of hydrazine; however, persistence of O6-methylguanine in liver DNA following hydrazine administration was longer in hamster than in rat liver DNA. Administration of hydrazine sulfate in the drinking water of hamsters over a 9-week period resulted in accumulation of both 7-methylguanine and O6-methylguanine in liver DNA to the extent that about 2 out of every 10,000 guanine bases were methylated at each position on the base. Diethyl maleate pretreatment of hamsters depleted liver stores of glutathione and blocked DNA methylation in hydrazine-treated animals; however, buthionine sulfoximine, which also depleted glutathione stores, had no effect on the DNA methylation response to hydrazine poisoning in Sprague-Dawley rats. Hydrazine administration to hamsters and Swiss Webster mice resulted in more 7-methylguanine and O6-methylguanine in liver DNA than did administration of monomethylhydrazine, a proposed intermediate in the hydrazine-DNA methylation response. Even at the highest feasible dose of monomethylhydrazine to hamsters, no methylguanines could be detected, while these aberrant bases were readily quantifiable following hydrazine administration; thus, no evidence was obtained to support the proposal that monomethylhydrazine is an important intermediate in the methylation of DNA guanine in hydrazine-treated animals.

Hydrazine administration to rats and mice results in a rapid methionine-mediated methylation of nucleophilic sites in liver DNA similar to sites alkylated by chemical carcinogens. The hydrazine-provoked methylation is only weakly dose-dependent and active for less than 20 hours following a single administration of toxicant. The methylating agent in poisoned animals appears to be S-adenosylmethionine and not a methylation product of hydrazine itself (namely monomethylhydrazine). Ethionine, an antimetabolite of methionine, when given shortly before hydrazine, inhibits DNA methylation; however, if the ethionine precedes hydrazine by several hours, DNA methylation is enhanced. Treatment of rats with small doses of ethionine soon after a large dose of hydrazine results in ethylation of DNA, an observation not expected for the amount of ethionine administered. Administration of other hepatotoxins, such as carbon tetrachloride or thioacetamide, also results in methionine-mediated methylation of liver DNA in rats. Administration of doses of as little as 100 mg/kg naphthalene, a volatile hydrocarbon in shale oil, results in severe bronchiolar epithelial cell necrosis in mice. This toxicity is prevented by pretreatment with an inhibitor of cytochrome P-450 and is increased substantially by pretreatment with a depletor of reduced glutathione.

Co-administration of an LDO.01 dose of hydrazine with 14C- methylmethionine to rats or mice results in the methylation at the 7-position of guanine in liver DNA; in the mouse, but not in the rat, methylation at the O6 - position of guanine in liver DNA also occurs. Methylation of liver DNA in only the 7-position of guanine occurs in rats and mice treated with MMH and to a much lesser extent with UDMH. Carbon dioxide production from MMH and UDMH is most active in rat liver, less active in kidney and colon and only slightly active in lung; carbon dioxide production from MMH is 1.4 to 5.5 times greater than for UDMH.

The relationship between oxidative metabolism and acute toxicity of dimethylnitrosamine (DMN) was examined in neonatal and adult rats to take advantage of developmental changes in activity of the metabolizing enzymes. The objective was to determine the extent to which CO2 production from DMN is correlated with toxicity. Neonatal rat liver and kidney demonstrated the ability to metabolize DMN. This ability progressed to a maximum activity in liver between the 5th and 21st d of age and in kidney between the 15th and 21st d of age. After weaning, the activity of DMN oxidation decreased with age in both tissues. Evidence is also presented to suggest that more than one enzyme may be responsible for the oxidation of DMN to CO2 and that the predominance of individual enzymes varies as the neonate develops. Estimates of LD50 values, used to quantitate the acute toxicity of DMN at various ages, suggest that the rat is most sensitive to DMN toxicity at 5 d of age; however, conversion of DMN to CO2, both in vitro and in vivo, was not well correlated with acute toxicity.

Oral administration of the inorganic hepatotoxin, hydrazine, to male Fischer 344 or Sprague Dawley rats results in the endogenous methylation of liver DNA at the 7- and O6-positions of guanine. At doses below the LD50 (45, 60 or 75 mg hydrazine/kg body weight), methylation levels varied little and averaged about 500micromol 7-methylguanine/mol guanine and 20 micromol O6- methylguanine/mol guanine. At 90 mg hydrazine/kg body weight (approximately the 7-day LD50) the methylation levels were 869 micromol 7-methylguanine and 58 micromol O6-methylguanine/mol guanine 24 hours after toxicant administration. A single dose of 3 mg hydrazine/kg body weight did not result in detectable levels of methylguanines in liver DNA; however, after three or four daily administrations of hydrazine at this dose, liver damage was evident, and liver DNA contained 50-100 micromol 7-methylguanine/mol guanine, about the limit of analytical detection. Following a single oral administration of 90 mg hydrazine/ kg body weight, liver DNA guanine rapidly became methylated. The time for half- maximum alkylation at 7-guanine was 30 minutes and at O6-guanine, 45 minutes. The rates of removal of these methylated bases were consistent with published values from experiments using methylating carcinogens and with values obtained in this laboratory with the model compound, 1,2-dimethylhydrazine.

The effect of CCl4 pretreatment (dose range from 0.5 to 2.0 ml/kg body weight) on the pathways of dimethylnitrosamine (DMN) metabolism was investigated. The oxidative-N-demethylation by the enzymes, DMN-demethylase I and II operating at low (4 mM) and high (200 mM) substrate concentration, respectively, was greatly reduced in a dose-dependent manner. In addition, the generation of an electrophilic intermediate capable of methylating DNA, specifically at the N-7 and O-6 positions of guanine, was completely inhibited by CCl4 (0.5 ml/kg body weight) pretreatment. When indole feeding (1% in the diet for 8 days prior to CCl4 administration) was employed as a means to enhance DMN-demethylase activities, it was found that the reduction of DMN-demethylase activities was more pronounced in these rats than in the controls. In agreement with earlier findings, 7-methylguanine and O6-methylguanine were not detectable in the CCl4 pretreated group. These results suggest that CCl4 exerts a strong inhibitory action on hepatic DMN metabolism, in particular on the pathway leading to alkylation of DNA guanine. This phenomenon may explain the protective role of CCl4 against DMN-induced hepatotoxicity and perhaps, carcinogenicity, believed to be closely associated with the abnormal modifications of DNA.

Hydrazine induces methylation of target-organ DNA guanine; the methylation mechanism was proposed to involve reaction of hydrazine with endogenous formaldehyde. One possible condensation product of hydrazine and formaldehyde is tetraformyltrisazine (TFT). TFT administered to Sprague-Dawley rats produced 7-methylguanine and O6-methylguanine in liver DNA at rates of formation and times to maximal methylguanine levels similar to those observed after hydrazine administration. TFT administration, however, resulted in greater amounts of methylguanines than did hydrazine on a molar basis, suggesting that TFT is perhaps a more proximal intermediate in hydrazine-induced methylation. The metabolic activation of TFT and hydrazine-plus-formaldehyde to methylating intermediates was detectable in in vitro systems containing as little as 0.2 mM TFT or 1 mM hydrazine-plus-formaldehyde, the lowest concentrations yet tested. The other major condensation product of hydrazine and formaldehyde, formalazine, also methylated liver DNA in vivo, but this polymer forms under conditions that would not be expected under in vivo administration of hydrazine. A novel pathway is proposed for the generation of a carbocation in hepatocytes exposed to hydrazine, consisting of condensation of hydrazine and formaldehyde to form TFT or formaldazine and/or formaldehyde hydroxymethylhydrazone and involves methylazomethanol as an intermediate.

Chemical analyses for aflatoxins were performed on autopsy specimens from 23 Thai children who died with acute encephalopathy and fatty degeneration of the viscera (EFDV) and from 15 children and adolescents who died from unrelated causes. Aflatoxin B1 was found in one or more specimens from 22 of the 23 EFDV cases. The highest levels detected were 93 μg B1/kg in a liver specimen, 123 μg/kg in stool, 127 μg/kg in stomach and intestinal contents and 8 μg/ml in bile. Trace amounts were detected also in brain, kidney and urine. Aflatoxin B2 was indicated, but not confirmed, in 12 specimens of brain, liver, kidney, stool and stomach and intestinal contents. A blue fluorescent spot with the chromatographic properties of aflatoxin M1 was found in trace amounts in two urine specimens and a spot similar to that of aflatoxin B2 was found in another. None of the urine specimens from healthy control children contained any of the aflatoxins, but very small amounts of aflatoxin B1 (1–4 μg/kg tissue) could be demonstrated in some autopsy specimens from 11 of the 15 control subjects.

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [14C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of 14C from l-[U−14C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.