C8-Guanine modifications: effect on Z-DNA formation and its role in cancer.

Base modifications are known to affect the structure and function of DNA. C8-guanine adducts from various carcinogenic compounds have been shown to be potent Z-DNA inducers. Hence, it has been hypothesized that Z-DNA plays a role in cancer and other genetic diseases. In this comprehensive review, Z-DNA and the effect of prevalent C8-guanine adducts on the B-Z transition are addressed. The discoveries of Z-DNA binding proteins including ADAR1, E3L, DLM1, and PKZ have suggested the relevance of Z-DNA in living systems. In addition, increasing evidence on the Z-DNA connection to gene transcription and inhibition reveals potential biological functions of the left-handed DNA. Finally, C8-guanine adducts that promote Z-DNA formation can be used as a tool to explore the Z-DNA function and its role in carcinogenesis.

In vitro reaction of barbiturates with formaldehyde.

Barbiturates are widely used as sedatives, hypnotics, and antiepileptics, and, when coupled with their narrow therapeutic index, the probability that their use will result in accidental or intentional death is significant. When barbiturates are implicated in a murder or suicide, analysis for their presence is often required. Under certain conditions, barbiturates are quite stable, but conditions found in vivo immediately after death or after embalming may promote barbiturate decomposition. If extensive decomposition occurs, analysis for them may be difficult or impossible. Here, the stability of three representative barbiturates, under conditions that model those likely to prevail in vivo shortly after death and after embalming, have been studied. Solutions of phenobarbital were found to slowly decompose in water over the pH range of approximately 3.5 to 9.5. More rapid decomposition occurred at higher pH, and 2-phenylbutyric acid was the main decomposition product. Formaldehyde (5-20%) accelerated the decomposition rate 3-10-fold such that phenobarbital decomposition could be complete after 30 days. In contrast, pentobarbital decomposed roughly 10 times more slowly and secobarbital did not detectably decompose under any of the conditions studied. Thus, certain barbiturates may partially or completely decompose in vivo after death, especially after embalming, and thus analysis for them may lead to false negatives. However, this work shows that analysis for the parent barbiturate or its predicted decomposition product may provide data that will reduce the likelihood of false negatives.

Stability of benzodiazepines in formaldehyde solutions.

Benzodiazepine-type drugs are used in the treatment of a number of pathologic disorders, but they may be implicated in forensic toxicology cases because of their abuse potential. Occasionally, it becomes necessary to measure drug levels following exposure to formaldehyde (postembalming or after tissue storage) if drug involvement was not previously suspected. Virtually no information exists on the decomposition of benzodiazepines in the presence of formaldehyde (the active ingredient in many embalming fluids), yet formaldehyde is known to be highly reactive, particularly with nitrogen-containing compounds. In order to evaluate the effects of formaldehyde on benzodiazepines, 10 benzodiazepine drugs were exposed to various concentrations of formaldehyde and various pH conditions (to simulate potential postembalming conditions), and the decomposition of each drug was measured by high-performance liquid chromatography over a 30-day period. The decomposition rates of all but one of the benzodiazepines were accelerated (to differing degrees) by formaldehyde as compared to controls, and this decomposition was in several cases both pH and formaldehyde concentration dependent. Thus, forensic examiners must be particularly cautious when attempting to determine benzodiazepine concentrations postembalming because the compound may have reacted with formaldehyde to form other products not inherently obvious analytically. Determination of these reaction products will serve to provide alternate analytes, allowing for establishment of accurate conclusions during forensic analyses.

In vitro reaction of formaldehyde with fenfluramine: conversion to N-methyl fenfluramine.

Embalming is common, and it can create problems for the forensic scientist if a drug has been the cause death and this drug is also reactive toward the embalming fluid. Previous studies have focused on the tricyclic amines nortriptyline and desipramine. In the presence of formaldehyde, a typical component of embalming fluid, either of these two compounds can be rapidly converted to their methylated derivatives amitriptyline and imipramine, respectively. We have begun a larger project designed to determine the reactivity and reactions of a wide range of drugs with formaldehyde. We report here our results from fenfluramine, which, like the tricyclic amines, is reactive towards formaldehyde and is converted into its N-methyl derivative. The rate of conversion is dependent upon pH and formaldehyde concentration. Up to 100% conversion in 24 h was observed. In addition, we have also devised a simplified procedure for monitoring this process that may be useful for others working in this area. Finally, we note that the reactions of fenfluramine studied here and of amines in general with formaldehyde need to be considered when performing postmortem/postembalming forensic analysis.

Ethanol-mediated CYP1A1/2 induction in rat skeletal muscle tissue.

The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se.

Activation of AP-1 through the MAP kinase pathway: a potential mechanism of the carcinogenic effect of arenediazonium ions.

Arenediazonium ions such as those found in the common mushroom Agaricus bisporus have been convincingly demonstrated to be tumorigenic. The specific mechanism of their tumorigenicity remains unclear. It has been shown that arenediazonium ions can be metabolized to aryl radicals, and that reaction of these aryl radicals with DNA produces aryl adducts. These metabolic processes also produce the reactive oxygen species superoxide and hydroxyl radicals which have been implicated in AP-1 activation. To further investigate the mechanism of tumorigenesis by arenediazonium ions, we studied the effect of a representative arenediazonium ion on AP-1 activation and phosphorylation of the signal transduction proteins ERK1, ERK2, JNK, and p38 kinase, both in vitro and in vivo. We also identified the specific radicals produced by spin trapping and ESR analysis. Here, it was found that p-methylbenzenediazonium ion (2a) induced a 16-fold increase in the extent of AP-1 activation at micromolar concentrations, and that this increase coincided with phosphorylation of the signaling kinases ERK1 and -2 and p38 kinase, but not JNK, in JB6 mouse epithelial cells. In vivo studies using AP-1 luciferase reporter-bearing transgenic mice supported the increase in the extent of AP-1 activation in 2a-treated mice over controls, and showed that this effect was different in different tissue types. The antioxidant N-acetylcysteine (NAC), a general antioxidant, showed an inhibitory effect on 2a-mediated AP-1 induction, while aspirin, a hydroxyl radical scavenger, had no effect. Spin trapping studies showed that while NAC suppressed radical formation from 2a, aspirin did not alter radical production from 2a. It appears that 3a, a carbon-centered radical formed from 2a, is responsible for AP-1-induced activation, and therefore, radical species that are not oxygen-centered are also capable of inducing AP-1. These results represent a step toward understanding the mechanism of tumorigenicity of arenediazonium ions.

Reactive oxygen species in the aerobic decomposition of sodium hydroxymethanesulfinate.

Sodium hydroxymethanesulfinate, (HOCH2SO2Na, HMS) is relatively stable in aqueous alkaline environments, but rapidly decomposes in acidic medium to give a variety of products that include sulfur dioxide. A detailed kinetic and mechanistic study of the decomposition of HMS in slightly acidic medium has shown a process that produces dithionite, S2O2-4, which is preceded by an induction period which persists for as long as molecular oxygen is present in the reaction solution. The complete consumption of molecular oxygen is a prerequisite for the formation of S2O2-4. Among some of the intermediates detected in the decomposition of HMS is the sulfite radical, SO-3. Comparisons are made between the decomposition mechanisms of thiourea dioxide (aminoiminomethanesulfinic acid) and HMS.

Cancer induction studies using different administrations of benzenediazonium sulfate in mice.

Benzenediazonium sulfate (BD) was administered as 10 weekly subcutaneous injections at 25 micrograms/g b.w. and as 52 weekly oral gavages at 100 micrograms/g b.w. to Swiss mice, starting at 6 weeks of age. The subcutaneous administration induced tumors in the subcutis with an incidence of 8% in females. The oral treatment gave rise to lung tumors with incidences of 52% in females and 62% in males. In the untreated control female mice, no subcutaneous tissue tumor was observed, but the incidences of lung tumors were 28% in females and 38% in males. Histopathologically, the neoplasms were classified as fibrosarcomas of the subcutis and adenomas and adenocarcinomas of the lungs. In an earlier experiment, BD induced high incidences of subcutaneous tissue tumors in the same species when it was administered as 26 weekly subcutaneous injections at 10 micrograms/g. This indicates the length of treatment is paramount to the dose of carcinogen. The oral route, even though it was carcinogenic in the lungs, failed to elicit the development of cancer in the glandular stomach.

C8-Arylguanine and C8-aryladenine formation in calf thymus DNA from arenediazonium ions.

Arylhydrazides, arylhydrazines, and N-alkyl-N-arylnitrosamines are metabolized to arenediazonium ions which yield C8-arylpurine adducts in calf thymus and cellular DNA. The mechanism of adduct formation has not been fully elucidated. C8-Arylguanine adducts likely form from direct aryl radical (Ar*) addition to the C8 position of guanine. However, the amounts of C8-aryladenine adducts measured here are inconsistent with direct radical attack at the C8 position of adenine. An intermediate product, an aryltriazene, is likely formed which then decomposes to the C8-aryladenine adduct. We have demonstrated that N1-aryl-N3-purinyltriazene adducts are formed from a variety of para-substituted arenediazonium ions with adenine. Decomposition of the N1-aryl-N3-purinyltriazene, at high pH and elevated temperatures, has been shown to give C8-aryladenine derivatives, and a free radical mechanism for this process has been proposed. Here we show that this process can occur under physiological conditions and that the C8-aryladenine adduct can be quantitated by HPLC. ESR studies, in which DMPO was used as a spin trap, have been used to demonstrate the intermediacy of aryl radicals during the decomposition of the N1-aryl-N3-purinyltriazenes and to demonstrate that this process also occurs in calf thymus (ct) DNA treated with arenediazonium ions. These results suggest the involvement of an aryl radical in the formation of the observed DNA adducts. Finally, we have found that the treatment of ct DNA with arenediazonium ions produces a significant amount of depurination. Both the formation of C8-arylguanine and C8-aryladenine adducts and the generation of apurinic sites may contribute to the genotoxicity of arylhydrazides, arylhydrazines, N-alkyl-N-arylnitrosamines, and arenediazonium ions.

Carcinogenesis by benzenediazonium sulfate in mice.

Benzenediazonium sulfate (BD) was given to Swiss mice by 26 subcutaneous injections of 10 micrograms/g body weight at weekly intervals. The treatment gave rise to tumors of the subcutis. The tumor incidences in the treated groups were 42% in females and 26% in males. The corresponding tumor incidences in the untreated controls were 0% in females and 2% in males. Histopathologically, the neoplasms were classified as fibrosarcomas, rhabdomyosarcomas, and osteosarcomas of the subcutaneous tissue. BD is formed during the cytochrome P-450 catalyzed metabolism of the carcinogenic 1-(phenylazo)-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14), which was used as a coloring agent for food and other materials in several countries. Further, BD is a metabolic breakdown product of different classes of nitrogen-nitrogen bond- containing chemicals. BD is the fourth benzenediazonium salt found to be carcinogenic in this laboratory.

Cobalt-mediated generation of reactive oxygen species and its possible mechanism.

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.

Carcinogenesis studies with the lyophilized mushroom Agaricus bisporus in mice.

Continuous administration of 10, 5, and 2.5% lyophilized Agaricus bisporus (AB) mushroom in the diet of six-week-old, randomly bred Swiss mice for life induced tumors in the lungs, forestomach, glandular stomach, and ovaries in certain groups. Some of the tumor incidences were found to be statistically significant, although no dose-response relationship was established. Histopathologically, the neoplasms were classified as adenomas and adenocarcinomas of lungs, glandular stomach, and ovaries and squamous cell papillomas and carcinomas of the forestomach. AB given in both raw and baked forms induced tumors in the same species in earlier experiments. Since this fungus is consumed in lyophilized form to a certain degree in the United States, the results may carry practical significance.

Lack of carcinogenesis by the baked mushroom Agaricus bisporus in mice: different feeding regimen [corrected].

Agaricus bisporus, the cultivated mushroom of the western hemisphere, was baked at 220-230 degrees C for 10 minutes and subsequently fed to mice for 12 hours each day, five days each week throughout their life. After each feeding cycle, the animals received a well-balanced semisynthetic diet for 12 hours each day for five days plus the remaining two full days each week. The estimated average daily mushroom consumption per animal was 4.8 g for a female and 4.2 g for a male. Randomly bred Swiss mice, six weeks old at the start of the experiment, were used. In the baked mushroom-fed group, the incidences of tumors in the lungs, blood vessels, cecum, and colon increased when compared to the untreated controls. These increases were not, however, statistically significant. In another previous experiment, both the raw and the baked mushrooms, when used in different feeding regimens, induced statistically significant incidences of cancers in several organs of the mice. It is possible that the negative finding in the current study was due to insufficient mushroom consumption.

Carcinogenesis by the cultivated baked Agaricus bisporus mushroom in mice.

The mushroom of commerce in the Western hemisphere, Agaricus bisporus, was administered orally to Swiss mice that were 6 weeks old at the start of the experiment The mushrooms were baked at 220-230 degrees C for 10 min. Subsequently, the mushrooms were fed to the animals for 3 days and were followed by a semisynthetic diet for 4 days each week, for life. The treatment induced tumors in the forestomach, glandular stomach, duodenum, and ovaries in the following incidences: 20, 12, 14 and 12% in females and 16, 20, 4 and 0% in males. In the tissues of the untreated controls, only an ovarian tumor was found in a female. Histopathologically, the neoplasms were classified as squamous cell papillomas and carcinomas of the forestomach, and adenomas and adenocarcinomas of the glandular stomach, duodenum, and ovaries. Since Agaricus bisporus is mainly eaten in baked form in the United States, the findings may carry useful implications.

Aryl radical formation during the metabolism of arylhydrazines by microsomes.

Many arylhydrazines are genotoxins, although the mechanism of their genotoxicity is unknown. Previous studies have shown that arylhydrazines are metabolized to arenediazonium ions, which produce C8-arylguanine adducts in DNA suggesting the intermediacy of an aryl radical. Here we have looked for the formation of aryl radicals from arylhydrazines and microsomes by ESR spin trapping. Only hydroxyl radicals are trapped upon incubation of p-methylphenylhydrazine with rat liver microsomes and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, hydroxyl and aryl radicals were trapped upon incubation of p-(methoxymethyl)phenylhydrazine with rat liver microsomes. Evidence for hydroperoxyl radical formation was also obtained. In contrast, when either of these substrates was incubated with microsomes from C5O cells, aryl and hydroxyl radicals were trapped. The ESR signal intensity of the spin-trapped aryl radicals parallels the extent of C8-arylguanine formation in DNA, and therefore, the aryl radical is likely the intermediate responsible for C8-arylguanine adduct formation. Aryl radicals and C8-arylguanine adducts may be related to the genotoxicity of arylhydrazines and related compounds that are oxidatively metabolized to arenediazonium ions, the precursor to aryl radicals, including arylalkyl nitrosamines, arylazo compounds, and triazenes.

8-Arylguanine adducts from arenediazonium ions and DNA.

Arenediazonium ions (ArN2+) are genotoxic though the source of their genotoxicity is unknown. The present studies were undertaken to determine if reductive decomposition of ArN2+ to aryl radicals (Ar) in the presence of calf thymus DNA (ctDNA) or in cells results in the formation of DNA adducts. We found that when arenediazonium ions of the general structure p-X-ArN2+ (X = CH3, CH2OCH3, CH2OH) are allowed to react with ctDNA or incubated with cells under conditions that produce p-X-Ar, DNA adducts are formed with guanine. The structure of the adduct is the C8-substitution product derived from guanine and p-X-Ar. Formation of p-X-Ar was determined by ESR spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The extent of C8-arylguanine adduction was measured by high performance liquid chromatography (HPLC) analysis of the DNA hydrosylate and comparison with authentic synthetic standards. The C8-arylguanine adducts observed to form may be important in regard to the genotoxicity of ArN2+, though other DNA adducts such as the N6-triazene of adenine or C8-aryladenine adducts can form. Finally, though the formation of C8-arylguanine adducts from arenediazonium ions has been proposed, this is the first report demonstrating their formation in DNA.

Vitamin B6 and cancer: synthesis and occurrence of adenosine-N6-diethylthioether-N-pyridoximine-5'-phosphate, a circulating human tumor marker.

In the course of studies aimed at deciphering the metabolic transformations of [3,4-14C] and [3H]C6-pyridoxine hydrochloride by tumor-bearing rats and tumor cells in culture, biosynthesis of a novel labeled product was observed. Its production began with the onset of tumor growth and increased as cell proliferation increased. Chemical, enzymatic, precursor labeling, and analytical tests on the isolated product indicated this product as adenosine-N6-diethylthioether-N-pyridoximine-5'-phosphate (compound 1). In confirmation, the chemical synthesis and characterization of compound 1 are presented in this study. In addition, blood samples from 28 normal subjects, 28 cancer patients with different malignancies, and 39 patients with a variety of other than cancer ailments were screened for compound 1 on a blind basis using reverse phase ion-paired high-performance liquid chromatography. The results show that the level of the vitamin B6 conjugate in the circulation of control subjects, cancer patients in remission, and patients with other diseases was only minimal. Cancer patients with active disease had 3-4-fold higher levels (P < 0.00001). Our results also confirm previous findings regarding the structure of compound 1 and show its potential value as a circulating human tumor marker that could be successfully used for cancer detection.

The use of 2-hydroperoxytetrahydrofuran as a reagent to sequence cytosine and to probe non-Watson-Crick DNA structures.

2-Hydroperoxytetrahydrofuran (THF-OOH) can be employed to sequence cytosine (C) and to probe for non-canonical DNA structures involving C. Using 32P-labeled oligomers and a DNA restriction fragment, it is demonstrated that THF-OOH has a strong preference for Cs in single-stranded (s-s) DNA regions, and in bulges, loops and mismatches. The reactivity of C is diminished below pH 6.0, but is not affected by substitution of 5-methylcytosine. To demonstrate the utility of the reagent, it is directly compared to methoxylamine and chloroacetaldehyde, two other reagents commonly used to chemically probe C residues in non-Watson-Crick DNA structures.

Gyromitra esculenta mushroom: a comparative assessment of its carcinogenic potency.

A comparative assessment is performed on one of the false morel mushrooms, Gyromitra esculenta, including the amounts of some hydrazines present in this fungus, the cancer-inducing doses of these chemicals or the mushroom used in animal experiments, the total amounts of the hydrazines or mushroom needed to induce neoplasms in mice and the estimated total amounts of hydrazines or mushroom needed to induce cancer in humans. When one compares the estimated amounts of hydrazines required to induce cancer with the amount of raw Gyromitra esculenta needed to yield a similar effect, it becomes clear that to date 37 percent of the carcinogenic ingredients of this fungus have been identified.

Generation of thiyl and ascorbyl radicals in the reaction of peroxynitrite with thiols and ascorbate at physiological pH.

Electron spin resonance (ESR) spin trapping was utilized to investigate the reaction of peroxynitrite with thiols and ascorbate at physiological pH. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The reaction of peroxynitrite with DMPO generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX). Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Thus, the spin trapping measurements provided no evidence for hydroxyl (.OH) radical generation in peroxynitrite decomposition at physiological pH. Thiols (glutathione, cysteine, and penicillamine) and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The one-electron oxidation of thiols by peroxynitrite may be one of the important mechanisms for peroxynitrite-induced toxicity and ascorbate may provide a detoxification pathway.