Locating nucleobase lesions within DNA sequences by MALDI-TOF mass spectral analysis of exonuclease ladders.

The location of carcinogen-modified nucleobases (DNA adducts) within DNA sequences is a critical factor affecting their promutagenic properties and persistence in DNA. We now report the use of controlled exonuclease digestion followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly map modified nucleobases within DNA. The DNA sequence is determined by mass spectral analysis of the DNA ladders produced by sequential removal of nucleotides with either 5'-->3' or 3'-->5' exonuclease. Individual mononucleotides are identified from the mass differences between adjacent peaks corresponding to singly charged ions of the products of enzymatic cleavage. Chemically modified nucleotides are detected and identified by their molecular weight. The resolution and mass accuracy of this approach are sufficient to identify nucleobase modifications differing in mass by as little as 2 Da. No a priori information on the DNA sequence or adduct type is required. We demonstrate the general applicability of this method by sequencing synthetic oligonucleotides containing a range of nucleobase modifications: O(6)-methylguanine, peroxynitrite-induced oxidative lesions (oxaluric acid, oxazolone, cyanuric acid), and the N(2)-guanine adduct of (+,-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydribenzo[a]pyrene. Sequence information is also obtained for DNA oligodeoxynucleotides containing O(6)-pyridyloxobutylguanine, despite the ability of this lesion to block 3'-phosphodiesterase.

The Biosure Study: influence of composition of diet and food consumption on longevity, degenerative diseases and neoplasia in Wistar rats studied for up to 30 months post weaning.

The 1200-rat Biosure Study had six interrelated aims: (1) To see whether dietary restriction (80% ad lib.) reduces the age-standardized incidence of fatal or potentially fatal neoplasia before the age of 30 months. (2) To see whether the beneficial effects of diet restriction can be achieved by (a) limiting the daily period of access to food to 6 hr, or by (b) limiting the energy value of the diet. (3) To see whether reduced calorie intake between weaning and age 4 months influences survival and/or incidence of non-neoplastic and neoplastic diseases. (4) To compare effects of food consumption, energy intake and protein intake on survival and disease. (5) To study the relationships between body weight at different ages with eventual survival and disease incidence. (6) To provide a database for studying relationships between various in-life measurements and eventual survival and disease incidence in individual animals. Twelve groups of SKF Wistar rats consisting of 50 animals of each sex were fed according to different dietary regimens from when they were weaned at the age of 3 wk until they died, or had to be killed because they were sick, or until the experiment was terminated at 30 months. For five of the 12 dietary regimens, satellite groups consisting of 30 animals per sex were maintained in parallel and used to supply information on the effect of diet on circulating hormone levels during the course of the study. During the 13 wk post weaning a Standard Breeder diet (SB) was provided either ad lib. (four groups), 80% ad lib. (three groups), or with access to food limited to 6 hr per day (one group). During this same period two other groups were fed a Low Nutrient Breeder diet (LB) ad lib. A further group was fed a Low Nutrient Maintenance (high fibre) diet (LM) ad lib. Finally, one group was fed the high protein Porton Rat diet (PR) ad lib.(ABSTRACT TRUNCATED AT 400 WORDS)

Type 2 cryoglobulinemia and hepatitis C virus: its recognition and treatment.

Type 2 mixed cryoglobulinemia is a relatively common although rarely recognized consequence of chronic hepatitis C virus infection. Its detection should be pursued in individuals with lower extremity vasculitis which occurs in association with other signs of systemic disease such as proteinuria or a peripheral neuropathy. Importantly, HCV-associated cryoglobulinemia can occur in individuals with clinical evidence for cryoglobulinemia but without any evidence of detectable liver injury. Two cases recently seen in Oklahoma demonstrating these points are reported.

Risks of premature death and cancer predicted by body weight in early adult life.

In a study of 30 months duration, involving 600 male and 600 female Wistar rats fed on 12 different diets/dietary regimes, none of which involved deliberate exposure to any known genotoxic carcinogen, highly significant between-group differences were observed in survival and incidence of various neoplastic and non-neoplastic diseases. A full report of the findings is being prepared. Here we report that, irrespective of diet or dietary regime, there were highly significant correlations of body weight at 29 weeks of age with premature death (P less than 0.0001 in both males and females), with development of benign or malignant neoplasm of any site (P less than 0.0001 in males and P less than 0.01 in females) and with development of malignant neoplasm at any site (P less than 0.0001 for sexes combined). Numerous kinds of neoplasm contributed to these overall correlations. The most significant were pituitary tumour (P less than 0.0001), mammary gland tumour (P less than 0.0001), squamous or anaplastic carcinoma of the jaw (P less than 0.001), and subcutaneous mesodermal tumours (P less than 0.05). The 20% of rats that were heaviest at 29 weeks were more than twice as likely to die prematurely than the lightest 20% (2.56 times--males, and 2.11 times--females), and almost twice as likely to develop a malignant tumour (1.87 times for the sexes combined). These findings have important implications for the design and interpretation of carcinogenicity tests in rodents and of laboratory and human studies of relationships between diet, ageing-related degenerative diseases, and cancer.

Effects of amino acid infusions on cell metabolism in hemodialyzed patients with uremia.

Eighteen uremic patients received i.v. infusions of a mixture of amino acids three times per week for 3 months in an effort to improve cellular amino acid imbalance and related cell bioactivities. They were a subset of 42 uremic patients stabilized by maintenance hemodialysis and were characterized by reduced cellular levels of threonine, isoleucine, methionine, and ornithine, with increased levels of aspartate, glycine, arginine, tyrosine, and phenylalanine. Protein synthesis (3H-leucine incorporation), energy level [energy charge = (ATP + 1/2 ADP)-(ATP + ADP + AMP)], and activity of the rate-limiting glycolytic enzyme pyruvate kinase were reduced in these patients compared with 32 control normal subjects. The circulating leukocyte (88 +/- 5% granulocytes) was used as a cell model. Previously, multiple regression best-subset analysis showed that a combination of the cell levels of aspartate, valine, isoleucine, ornithine, lysine, and tryptophan could "explain" 40% of the variance in protein synthesis in these patients. Similarly, a combination of the levels of aspartate, glutamic acid, glycine, ornithine, and arginine were "predictive" of the level of energy charge. We hypothesized that protein synthesis and energy level would be improved if the amino acid infusions normalized the intracellular levels of those amino acids that were predictive of the bioactivities. After 3 months of amino acid infusions, levels of the predictive intracellular amino acids were not improved, but deviated further from baseline values. Failure to detect significant improvement in the cell bioactivities is attributed to inability to correct the imbalance in the intracellular amino acid pool.

Toxicological evaluation of cyclosporin A.

Toxicological studies in a variety of different animal species have shown that cyclosporin A (CS-A) is a unique immunosuppressant, since it does not cause myelotoxic, teratogenic, mutagenic, or carcinogenic effects. This is undoubtedly due to the fact CS-A produces its immunosuppressive effects by a specific action on T lymphocytes rather than by a cytotoxic mechanism. The incidence of acute inflammatory lesions was also not significantly higher in treated animals than in controls. Clear-cut organ toxicity only occurred in rats and cynomolgus monkeys, where at high doses nephro- and hepatotoxicity were observed.

Mutagenicity evaluation of amino-oxazoline derivatives using in vitro and in vivo short-term tests.

During routine investigation of potential drugs it was found that 1-methyl-4-(2-oxazoline-2-ylamino)-indazole (1) compound 1 was mutagenic for Salmonella typhimurium TA1535 and TA100. The genotoxicity of compound 1 was confirmed by the following in vitro test systems: V79 Chinese hamster cells (HGPRT-locus), Saccharomyces cerevisiae D7 (mitotic gene conversion, mutations, aberrations), and Escherichia coli (rec-assay). On the other hand, when compound 1 was tested in vivo (micronucleus test in mice and sister chromatid exchange in Chinese hamsters) it did not show evidence of genotoxic activity. In order to study structure/activity relationships, different analogues of compound 1 were tested in Salmonella typhimurium TA1535. The tests showed that (1) most 2-amino-oxazolines were mutagenic for the test organism; (2) the 2-amino-imidazoline and 2-amino-thiazolidine derivatives tested were not mutagenic; (3) substituents bound to the extranuclear nitrogen of the 2-aminooxazoline ring had only a weak influence on the mutagenic potential; and (4) methylation of the oxazoline ring, most conspicuously at the carbon in position 5, strongly reduced the mutagenic effect. From these observations it is concluded that the reaction of nucleophilic DNA sites with the most electrophilic site of the oxazoline ring, ie, the carbon in position 5, is responsible for the genotoxicity of amino-oxazoline compounds. Due to the genotoxicity observed in these in vitro tests this class of compounds was no longer developed, but dropped, even without performing a long-term carcinogenicity study or considering the negative in vivo findings.

Critical appraisal of the 'mouse testicular DNA-synthesis inhibition test' for the detection of mutagens and carcinogens.

A short test (DSI test) in vivo for the identification of environmental mutagens and carcinogens was recently published by Friedman and Staub (1976), and Seiler (1977). The test is based on the measurements of [3H]thymidine incorporation into murine testicular DNA. This incorporation was inhibited by a number of mutagens and carcinogens when given at high doses. However, under such conditions, many chemicals are known to cause hypothermia in mice, which may itself lead to a reduced [3H]thymidine incorporation. We therefore investigated the effect of a few non-mutagenic and mutagenic test substances on the incorporation of [3H]thymidine into testicular DNA and on changes in body temperature. We also studied the effect of reduced testicular temperature, caused by physical means, on the incorporation of [3H]thymidine into DNA. 3 mutagens out of 4, and 6 non-mutagens out of 6, caused hypothermia in mice, and at the same time inhibited [3H]thymidine incorporation. This parameter also changed as the testicular temperature was reduced, indicating an obvious correlation between the 2 effects. Furthermore, we found that, with some of the test substances, a reduction of [3H]thymidine incorporation into DNA can be compensated under isothermal conditions. We believe that the DSI test is not reliable as a screening system for the identification of potential mutagens and cancerogens because of the unspecificity of the parameter measured.

Toxicological evaluation of fluproquazone.

The toxicological characteristics of 4-(p-fluorophenyl-1-isopropyl-7-methyl-2-(1H)quinazolinone (fluproquazone), an analgesic with distinct antiinflammatory properties, were evaluated in acute and chronic toxicity studies as well as in reproduction toxicity, carcinogenicity and mutagenicity studies. The following overall results were obtained: The acute oral toxicity in mice, rats, and rabbits is of low order. In the chronic oral studies fluproquazone was generally well tolerated when given to rats and dogs for 13 weeks, to dogs and monkeys for 52 weeks, to mice for 78 weeks and to rats for 104 weeks. In particular, there was no indication of gastrointestinal irritations or lesions in any of these studies. The results of the studies in dogs and rats showed the major target organs for the toxicity of fluproquazone to be the liver and kidney, where mild, reversible changes were observed. These findings were considerably less severe than those found with several other antiphlogistic-analgesic compounds. In the reproduction toxicity studies, the only drug-related effects seen in experiments on female fertility or peri- and postnatal development in rats were a prolongation of pregnancy and an impairment of delivery leading to an increased perinatal mortality. These findings may be related to an inhibition of prostaglandin synthesis by fluproquazone. Similar effects are known to occur after administration of other inhibitors of prostaglandin synthesis. The oral teratological studies in rats and rabbits did not reveal any embryolethal or teratogenic effects. The drug had no mutagenic effects in either the micronucleus test and the dominant-lethal test using mice, or in the Ames-Test using Salmonella typhimurium. The carcinogenicity studies showed that fluproquazone has no carcinogenic potential in rats and mice.

Mutagenicity test systems for the detection of chromosome aberrations in vivo.

In mutagenicity testing of pharmaceuticals it is advisable, whenever possible, to use in vivo mammalian systems where the pharmacokinetic properties of a drug would be more or less similar to those in man. However, although there are a number of different genetic endpoints which are important in mutagen testing, sufficiently validated, practical and well documented in vivo test systems exist only for structural chromosome aberrations. This paper provides an overview of existing in vivo test systems for the detection of chromosome aberrations. Emphasis has been laid on the functions, advantages and limitations of the three tests recommended in the guidelines of the European Community; first, the metaphase analysis assay, and, as alternatives, the micronucleus test and the dominant lethal test. These three tests are then also discussed from the viewpoint of our own practical experience.

Relief of idiopathic generalized pruritus in dialysis patients treated with activated oral charcoal.

The effect of oral charcoal on idiopathic generalized pruritus in 11 stable patients undergoing maintenance hemodialysis was compared to that of placebo dextrose in a controlled, double-blind, cross-over study. Contrasted to placebo, charcoal, 6 g daily for 8 weeks, relieved pruritus subjectively in all but one patient (P = 0.01). Symptomatic relief from pruritus coincided with objective resolutions of active, scratch-induced skin lesions (P = 0.03). No significant alterations were noted in the serum concentrations of standard laboratory variables, including lipids, alkaline phosphatase, phosphorus, or calcium, during treatment with either charcoal or placebo. No adverse effects from the charcoal were noted during the study.