Mutation of V79 cells by N-dialkylnitrosamines after activation by hamster pancreas duct cells.

Pancreas duct epithelial cells (DEC), isolated from hamsters and cultured for up to 25 days, were able to metabolize N-nitrosobis(2-oxopropyl)amine (BOP) to species that were mutagenic in V79 cells. There was no decline in the nitrosamine-activating ability of DEC over the period of observation (25 d). DEC activated N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosodiethylamine (DEN), N-nitrosodimethylamine (DMN) and N-nitrosomethyl(2-oxopropyl)amine (MOP) and BOP in the same assay, although the mutation frequencies for BHP, DEN and DMN were barely different from that for the controls (4 +/- 1 mutants/10(6) cells). The mutation frequencies for a dose of 0.1 mM were BHP, 2 +/- 1; BOP, 113 +/- 7; DEN, 8 +/- 1; DMN, 5 +/- 2; and MOP, 18 +/- 3 (mutants/10(6) cells; means +/- SE). When hepatocytes were used the mutation frequencies were BHP, 3 +/- 1; BOP, 60 +/- 3; DEN, 8 +/- 2; DMN, 8 +/- 2; and MOP, 121 +/- 10. BOP was toxic to the DEC at doses above 0.1 mM. Experiments in which co-factors were omitted from the medium suggested that an isoform(s) of the cytochrome P-450 IIIA family was involved, directly or indirectly, in BOP activation.


An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds.

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.


An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. II. In vivo results for 36 compounds tested in the mouse.

The aim of this study was to further evaluate the E. coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs. The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E. coli K-12. After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host. A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties. A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay. Positive results were obtained for 23 compounds. Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative. The overall concordance between the assay and carcinogenicity was 72%. In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs. With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood. The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances. Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide. The substances negative in the E. coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide. It is concluded from this evaluation that the E. coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter. The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)


Genotoxic potential of the organochlorine insecticide lindane (gamma-BHC): an in vivo study in chicks.

The purpose of this work was to evaluate the genotoxic potential of lindane (gamma-isomer of benzene hexachloride (BHC)) in chicken in vivo tests: the bone marrow chromosome aberration and micronucleus tests. With the highest dose (100 mg/kg) a significant enhancement of chromosome aberrations was noticed after 24 and 48 h and with the second highest dose (75 mg/kg) after 24 h. A significant increase in the incidence of micronuclei in bone marrow cells was induced by all three doses (100, 75 and 50 mg/kg) given either intraperitoneally or orally while in peripheral erythrocytes only the two higher intraperitoneal doses (100 and 75 mg/kg) gave significant increases. On the basis of these results, lindane may be considered genotoxic in this test system and it is suggested that the chick in vivo system may be used as an alternative to a mammalian system for screening environmental chemicals for genotoxicity.


Use of a newly developed tester strain Salmonella typhimurium NM2009 for the study of metabolic activation of carcinogenic aromatic amines by rat liver microsomal cytochrome P-450 enzymes.

Using an O-acetyltransferase-overexpressing strain Salmonella typhimurium NM2009 we measured the activities for metabolic activation of several carcinogenic arylamines to genotoxic products by rat liver microsomal cytochrome P-450 enzymes, and compared them with the activities obtained in the original tester strain Salmonella typhimurium TA1535/pSK1002 or the O-acetyltransferase-defective strain Salmonella typhimurium NM2000. Since all of the tester strains had introduced the umuC'-'lacZ gene, we could detect the genotoxic activities by measuring bacterial beta-galactosidase activity resulting from the DNA damage. In the O-acetyltransferase-defective strain NM2000 most of the arylamines tested showed weak responses in inducing umu gene expression after metabolic activation by liver microsomes. The strain NM2009, on the other hand, was found to be highly sensitive towards a variety of aromatic amines, and these activities were greater than those seen in the original tester strain S. typhimurium TA1535/pSK1002. The chemicals which marked responses in strain NM2009 include 2-aminoanthracene, 6-aminochrysene, 2-aminofluorene, 2-acetylaminofluorene, 3-methoxy-4-aminoazobenzene, O-aminoazotoluene, Glu-P-1, Trp-P-2, A alpha C, MeA alpha C, MeIQ, MeIQx and IQ. Of these procarcinogens tested MeIQ, MeIQx and IQ also showed strong cytotoxic effects in S. typhimurium NM2009 after metabolic activation by liver microsomes. Only PhIP was the substrate showing similar responses in strains TA1535/pSK1002 and NM2009. The results with the reconstituted monooxygenase system containing purified cytochrome P-450 enzymes support the above findings obtained with the liver microsomal enzyme system. Thus, the usefulness of the newly developed strain NM2009 for the detection of reactive metabolites of several carcinogenic aromatic amines after metabolism by the liver microsomal cytochrome P-450-linked monooxygenase system has been ascertained.


A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.


Progressive loss of cytochrome c oxidase in the human extraocular muscles in ageing--a cytochemical-immunohistochemical study.

Cytochrome c oxidase (complex IV of the respiratory chain) was studied histochemically in autoptic human extraocular muscles (n = 135), revealing randomly distributed single fibers without enzyme activity. The enzyme defect was expressed in all the mitochondria of an involved fiber as evidenced by ultracytochemistry. Succinate dehydrogenase showed normal histochemical reactivity. The defects occurred already in the second decade and were regularly seen from the third decade on. The defect density (defects/mm2) increased from approx. 1/mm2 below the fifth decade to about 4/mm2 in advanced age (P = 0.000). The highest defect density was observed in the levator palpebrae muscle. On the whole, the defect density was about 5-6 times higher in the extraocular muscles than in the limb muscle, diaphragm and heart (Müller-Höcker, 1989, 1990). Immunocytochemical detection of cytochrome c oxidase showed that loss of cytochrome c oxidase activity was due to an almost complete absence of both nuclear and mitochondria subunits of the enzyme. The results document different organ and heterogenic cellular sensitivity to the age-related loss of cytochrome c oxidase. The loss of both mitochondrial and nuclear subunits indicates that nuclear factors are most probably involved in the decline of the respiratory chain function in senescence.


Respiratory chain failure in adult muscle fibres: relationship with ageing and possible implications for the neuronal pool.

A histochemical analysis of mitochondrial enzyme activity was carried out in 103 human diaphragmatic skeletal muscles from 49 subjects of different ages, obtained either at the time of abdominal surgery or at necropsy. Evidence of respiratory failure (cytochrome oxidase negativity) was seen in occasional fibres from the fourth decade on with an approximate 10-fold increase between the fourth and ninth decade (0.16% to 2.85%). A similar incidence of mitochondrial failure in CNS neurones to that documented in skeletal muscle could easily account for attrition of 25% of neurones over a 50-year period as reported in the literature. Possible theoretical relationships between morphological markers of mitochondrial failure and cell attrition are explored. While the projections from muscle to neurone are somewhat speculative, it is clear that if a similar extent of mitochondrial pathology exists in the brain to that documented in skeletal muscle, this could easily account for neuronal loss in the ageing brain.


Mitochondrial function in neurodegeneration and ageing.

The mitochondrial respiratory chain and oxidative phosphorylation system are responsible for the production of ATP by aerobic metabolism. Defects of the respiratory chain are increasingly recognised as important causes of human disease, and neurodegenerative disorders in particular. This article will seek to review the clinical and biochemical effects of respiratory chain defects, and summarise what is known about the molecular mechanisms that underlie them. Increasing age is also associated with a decline in mitochondrial function. The biochemical correlates of this dysfunction and the possible molecular defects that may cause it will also be reviewed.


Mitochondrial DNA alterations as ageing-associated molecular events.

Mitochondrial DNA (mtDNA) is a naked double-stranded circular extrachromosomal genetic element continuously exposed to the matrix that contains great amounts of reactive oxygen species and free radicals. The age-dependent decline in the capability and capacity of mitochondria to dispose these oxy-radicals will render mtDNA more vulnerable to mutations during the ageing process. During the past 3 years, more than 10 different types of deletions have been identified in the mtDNA of various tissues of old humans. Some of them were found only in a certain tissue but some others appeared in more than one organ or tissue. The 4977-bp deletion is the most prevalent and abundant one among these deletions. Skeletal muscle is the target tissue of most ageing-associated mtDNA deletions and has often been found to carry multiple deletions. The onset age of the various deletions in mtDNA varies greatly with individual and type of the deletion. The 4977-bp deletion has been independently demonstrated to occur in the mtDNA of various tissues of the human in the early third decade of life. However, the 7436-bp deletion was only detected in the heart mtDNA of human subjects in their late thirties. The others appeared only in older humans over 40 years old. No apparent sex difference was found in the onset age of these ageing-associated mtDNA deletions. The various ageing-associated deletions could be classified into two groups. Most of the deletions belong to the first group, in which the 5'- and 3'-end breakpoints of the deletion are flanked by 4-bp or longer direct repeats. The deletion in the second group occurs less frequently and shows no distinct repeat sequences flanking the deletion sites. These two groups of mtDNA deletions may occur by different mechanisms. The first group is most probably caused by internal recombination or slippage mispairing during replication of mtDNA by the D-loop mechanism. The deleted mtDNA and the deleted DNA fragment may be further degraded or escape from the mitochondria and get translocated into the nucleus. The latter route has been substantiated by many observations of inserted mtDNA sequences in the nuclear DNA. Thus, the fragments of migrating mtDNA may change the information content and expression level of certain nuclear genes and thereby promote the ageing process or cause cancer. Similar ageing-associated alterations of mtDNA have also been observed in aged animals and plants. I suggest that mtDNA deletions and other mutations to be discovered are molecular events generally associated with the ageing process.


Deleterious mitochondrial DNA mutations accumulate in aging human tissues.

This paper reviews the current state of knowledge of the contribution of mitochondrial DNA (mtDNA) mutations to the phenotype of aging. Its major focus is on the discovery of deletions of mtDNA which previously were thought to occur only in individuals with neuromuscular disease. One particular deletion (mtDNA4977) accumulates with age primarily in non-dividing cells such as muscle and brain of normal individuals. The level of the deletion rises with age by more than 1000 fold in heart and brain and to a lesser extent in other tissues. In the brain, different regions have substantially different levels of the deletion. High levels of accumulation of the deletion in tissues are correlated with high oxygen consumption. We speculate that oxidative damage to mtDNA may be 'catastrophic'; mutations affecting mitochondrially encoded polypeptides involved in electron transport could increase free radical generation leading to more mtDNA damage.


Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease.

The role of somatic mitochondrial DNA (mtDNA) damage in human aging and progressive diseases of oxidative phosphorylation (OXPHOS) was examined by quantitating the accumulation of mtDNA deletions in normal hearts and hearts with coronary atherosclerotic disease. In normal hearts, mtDNA deletions appeared after 40 and subsequently accumulated with age. The common 4977 nucleotide pair (np) deletion (mtDNA4977) reached a maximum of 0.007%, with the mtDNA7436 and mtDNA10,422 deletions appearing at the same time. In hearts deprived of mitochondrial substrates due to coronary artery disease, the level of the mtDNA4977 deletion was elevated 7-220-fold over age-matched controls, with the mtDNA7436 and mtDNA10,422 deletions increasing in parallel. This cumulative mtDNA damage was associated with a compensatory 3.5-fold induction of nuclear OXPHOS gene mRNA and regions of ischemic hearts subjected to the greatest work load (left ventricle) showed the greatest accumulation of mtDNA damage and OXPHOS gene induction. These observations support the hypothesis that mtDNA damage does accumulate with age and indicates that respiratory stress greatly elevates mitochondrial damage.


Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats.

In order to understand the cause of the reduced mitochondrial DNA transcription in heart and brain of senescent rat previously reported, we focused our attention on the content and structure of rat mitochondrial DNA in adult and senescent rats. The estimate of the mtDNA copy number in liver, heart and brain of adult and senescent rats showed that in all organs examined the senescent individuals have a mtDNA content higher than the adult counterparts. The analysis of mtDNA structural changes involved the search for point mutations and large deletions. As for the first case, the determination of the nucleotide sequence of many independent clones containing two mtDNA restriction fragments isolated from rat cerebral hemispheres did not show any sequence difference between adult and senescent individuals. However, analysis of mtDNA deletions by the polymerase chain reaction in liver and brain of adult and senescent rats identified a small population of mtDNA molecules harboring a deletion of 4834 bp. The estimate of the proportion of deleted molecules in the liver showed that they represent 0.02% and 0.0005% of total mtDNA in senescent and adult rat liver respectively. Therefore, a mtDNA deletion also accumulates in the rat during aging. This result supports the hypothesis of the accumulation of deleted mtDNA molecules in aging. However, the low percentage of deleted mtDNA molecules already found and the reversibility of the reduced mitochondrial DNA transcription in senescent rat raise doubts on the primary role of the irreversibly damaged mtDNA molecules in aging. Deleted mtDNA molecules along with changes caused by lipid peroxidation of mitochondrial membranes might contribute to the overall decline of mitochondrial function.


Mitochondrial DNA mutation and the ageing process: bioenergy and pharmacological intervention.

A comprehensive hypothesis concerning the contribution of mitochondrial DNA (mtDNA) mutations to the human ageing process is reviewed and the implications for cellular bioenergy loss and pharmacological therapy are considered. The central idea is that random mutations in the population of mtDNA molecules of each cell occur throughout life, and that this is a major contributor to the gradual loss of cellular bioenergy capacity within tissues and organs, associated with general senescence and diseases of ageing. An elaboration of four major aspects of the general proposition, together with relevant supporting data, is presented. (1) An extensive array of deletions in mtDNA of many tissues of humans and other mammals has been observed to occur in an age-related manner. (2) The preservation and selection of fully functional mtDNA molecules in the female germ line cells is proposed to occur via a human mtDNA cycle, in which selective amplification of a limited number of mtDNA templates occurs during oocyte development. This proposal explains the endowment of normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA molecules. The phenomena of maternal inheritance and rapid fixation of sequence variants of mtDNA in mammals, as well as selection of cells based on mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics result from accumulated mtDNA damage during ageing, representing different rates of cellular bioenergy loss within individual cells of a tissue. The random segregation of mtDNA during cell division will also further contribute to the tissue energy mosaic. Cells unable to meet their particular bioenergy demand will become non-functional, leading to cell death; the bioenergy threshold is different for the various cell types in the tissues of the body. (4) In order to bioenergetically resuscitate cells and tissues suffering from impaired mitochondrial functions as a result of the ageing process, we propose that redox compounds may be used therapeutically in the pharmacological configurations of a by-pass strategy or as a redox sink therapy. The role of these compounds is to maintain at least part of the mitochondrial respiratory chain function (by-pass) as well as to maintain adequate levels of cellular NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic pathway, with some contribution from mitochondrial oxidative phosphorylation.


An update on the mitochondrial-DNA mutation hypothesis of cell aging.

Our electron microscopic study of aging insects and mammals suggests that metazoan senescence is linked to a gradual process of mitochondrial breakdown (and lipofuscin accumulation) in fixed postmitotic cells. This led us to propose in the early 1980s an oxyradical-mitochondrial DNA damage hypothesis, according to which metazoan aging may be caused by mutation, inactivation or loss of the mitochondrial genome (mtDNA) in irreversibly differentiated cells. This extranuclear somatic gene mutation concept of aging is in agreement with the fact that mtDNA synthesis takes place at the inner mitochondrial membrane near the sites of formation of highly reactive oxygen species and their products. Mitochondrial DNA may be unable to counteract the damage inflicted by those by-products of respiration because, in contrast to the nuclear genome, it lacks excision and recombination repair. Since mtDNA contains the structural genes for 13 hydrophobic proteins of the respiratory chain and ATP synthase as well as mitochondrial rRNAs and tRNAs, damage to this organellar genome will decrease or prevent the 'rejuvenation' of the mitochondria through the process of macromolecular turnover and organelle fission. Thus deprived of the ability to regenerate their mitochondria, the fixed postmitotic cells will sustain a decrease in the number of functional organelles, with resulting decline in ATP production. At higher levels of biological organization, this will lead to a loss in the bioenergetic capacity of cells, with concomitant decreases in ATP dependent protein synthesis and specialized physiological function, thus paving the way for age related degenerative diseases. The above concept is supported by a wealth of recent observations confirming the genomic instability of mitochondria and suggesting that animal and human aging is accompanied by mtDNA deletions and other types of injury to the mitochondrial genome. Our hypothesis of mtDNA damage is integrated with the classic concepts of Weissman and Minot in order to provide a preliminary explanation of the evolutionary roots of aging and reconcile the programed and stochastic views of metazoan senescence.


Evidence for and against the causal involvement of mitochondrial DNA mutation in mammalian ageing.

Current experimental evidence on the role of mitochondrial DNA mutation in ageing is assessed alongside reports implicating other genetic and non-genetic causes, including inter-relationships between the mitochondrial and nuclear genomes and their potential effect on mitochondrial structure and function. The role of a 5-kb mtDNA deletion, identified as age-dependent in a variety of human and other mammalian species, is specifically evaluated in the context of its functional effect in mitotic and non-mitotic adult tissue. Downstream effects of mitochondrial decline are considered in terms of the maintenance of ATP production. Associated sequelae then are discussed specifically with reference to restrictions in the supply of ribose moieties for DNA and RNA synthesis, and to disruption of NADPH production and hence cellular anti-oxidant defences.


New evidence for the insertion of mitochondrial DNA into the human genome: significance for cancer and aging.

We have observed and characterized in detail two cases of mitochondrial DNA fragments which have inserted into the nucleus of HeLa cells. In one case three non-sequential but contiguous regions of mitochondrial DNA with 92% homology to human cytoplasmic mitochondrial DNA inserted into the nuclear genome. In the second case the mitochondrial DNA sequence encoding cytochrome c oxidase subunit III was contiguous with and 5' of exons 2 and 3 of the c-myc oncogene and the chimeric gene was transcribed. Models are presented that describe mechanisms for the transfer of mitochondrial DNA into the nucleus involving fragmentation of mitochondrial DNA through aging and/or oxidative damage, anomalous processing or escape of mitochondrial DNA and RNA fragments from autophagic vacuoles, and insertion of mitochondrial DNA sequences, in some instances after reverse transcription of mitochondrial RNA, into the nuclear genome.


Structural dynamics of the mitochondrial compartment.

The metabolic activities of mitochondria have been extensively characterized. However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism. There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions. Segregation and recombination of mitochondrial genomes are also processes dependent upon the dynamic nature of the mitochondrial compartment. These regulatory and structural aspects of mitochondrial compartment dynamics will play an important role in the analysis of mitochondrial function and pathology.


Lipid peroxidation and mtDNA degeneration. A hypothesis.

End-products of lipid peroxidation accumulate during the life of somatic cells. It is hypothesized that genotoxic intermediates of lipid peroxidation may have a role in causing age-associated DNA mutations. Such mutations are likely to accrue in the mitochondrial genome because it, unlike nuclear DNA, is not protected by histones and repair systems. In addition, it is located near the mitochondrial membrane where lipid peroxidation can be initiated by free radicals produced by the mitochondrial electron transport system. This idea is supported by in vitro experiments which show that mitochondrial DNA is damaged when mitochondria undergo lipid peroxidation.


Reactive oxygen and DNA damage in mitochondria.

During the last decade the importance of reactive oxygen species as major contributors to various types of cancer, heart diseases, cataracts, Parkinson's and other degenerative diseases that come with age, and to natural aging has become apparent. Mitochondria are the most important intracellular source of reactive oxygen. Mitochondrial DNA is heavily damaged by reactive oxygen at the bases, as indicated by the high steady-state level of 8-hydroxydeoxyguanosine, the presence of which causes mispairing and point mutations. Mitochondrial DNA is also oxidatively fragmented to a certain extent. Conceivably, such fragmentation relates to deletions found in mitochondrial DNA. Point mutations and deletions have recently been shown to be etiologically linked to several human diseases and natural aging. Future studies should address the causal relationship between mitochondrial dysfunction, production of reactive oxygen species, and aging.


Free radical theory of aging.

Free radical reactions are ubiquitous in living things. Studies on the origin and evolution of life provide a reasonable explanation for the prominent presence of this unruly class of chemical reactions. These reactions have been implicated in aging. This phenomenon is the accumulation of changes responsible for the sequential alterations that accompany advancing age and the associated progressive increases in the chance of disease and death. Aging changes are attributed to the environment and disease, and to an inborn process, the aging process. The latter produces aging changes at an exponentially increasing rate with advancing age. Past improvements in general living conditions have decreased the chances for death so that they are now near limiting values in the developed countries. In these countries the intrinsic aging process is the major cause of disease and death after about age 28. The free radical theory of aging postulates that aging changes are caused by free radical reactions. The data supporting this theory indicate that average life expectancy at birth may be increased by 5 or more years, by nutritious low caloric diets supplemented with one or more free radical reaction inhibitors.


Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.


Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase.

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid peroxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47; P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 +/- 0.71 mumole MDA/g tissue) than at 2 months (5.69 +/- 0.90) and significantly lower at 28 months (5.13 +/- 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.


Mitochondrial production of pro-oxidants and cellular senescence.

Mitochondria are the major intracellular producers of O2- and H2O2. The level of oxidative stress in cells, as indicated by the in vivo exhalation of alkanes and the concentration of molecular products of oxy-radical reactions, increases during aging in mammals as well as insects. In this paper, we discuss the relationship between mitochondrial generation of O2- and H2O2, and the aging process. The rate of mitochondrial O2- and H2O2 generation increases with age in houseflies and the brain, heart and liver of rat. This rate has been found to correspond to the life expectancy of flies and to the maximum life span potential (MLSP) of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow. In contrast, the level of antioxidant defenses provided by activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione concentration neither uniformly declines with age nor corresponds to variations in MLSP of different mammalian species. It is argued that the rate of mitochondrial O2- and H2O2 generation rather than the antioxidant level may act as a longevity determinant.


Oxidative and other DNA damages as the basis of aging: a review.

DNA damages occur continuously in cells of living organisms. While most of these damages are repaired, some accumulate. In particular, there is evidence for DNA damage accumulation in non-dividing cells of mammals. These accumulated DNA damages probably interfere with RNA transcription. We consider that the decline in the ability of DNA to serve as a template for gene expression is the primary cause of aging. Oxidative DNA damages are among the best documented and prevalent DNA damages and are likely to be a prominent cause of aging.


DNA damage and repair in brain: relationship to aging.

The usefulness of conducting DNA damage and repair studies in a postmitotic tissue like brain is emphasized. We review studies that use brain as a tissue to test the validity of the DNA damage and repair hypothesis of aging. As far as the accumulation of age dependent DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting and equally divided. Possible reasons for this discrepancy are discussed. It is suggested that assessment of the repair capacity of neurons with respect to a specific type of damage in a specific gene might yield more definitive answers regarding the role of DNA repair potential in the aging process and as a longevity assurance system.


Oxidative damage to DNA in mammalian chromatin.

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivo by a variety of free radical-producing systems. Methodologies incorporating the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H2O2 and in chromatin of organs of animals treated with carcinogenic metal salts.


Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.


Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.


Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.


Interaction of singlet oxygen with DNA and biological consequences.

To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp). A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentified reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp. Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively. A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible. In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.


Hypothesis: a damaging role in aging for reactive protein oxidation products?

This paper discusses our knowledge of protein oxidation and its relationship to aging. It also outlines new observations from our laboratories concerning reactive species produced during protein oxidation, and proposes that these may inflict damage on other molecules, and hence contribute to the progression of aging. Whereas it has previously been difficult to see how relatively inert protein oxidation products could possibly have any causal role in aging, the detection of these novel reactive species implies a potentially significant role.


A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis.

Based on a series of experiments, using cultured postmitotic neonatal rat cardiac myocytes as a model system, we present a novel hypothesis of lipofuscin formation. This hypothesis proposes that lipofuscin is formed within secondary lysosomes due to an interplay of two processes, the production of partially reduced oxygen species by mitochondria and the autophagocytotic degradation within secondary lysosomes. Specifically, it is proposed that H2O2 generated by mitochondria and other organelles permeates into the lumen of secondary lysosomes, which contain iron derived from cellular structures undergoing intralysosomal degradation. The interaction between reactive ferrous iron and H2O2 results, via Fenton-type mechanisms, in the generation of hydroxyl free radicals (OH), inducing lipid peroxidation and eventually leading to intermolecular cross-linking and lipofuscin formation. Additionally, mitochondria undergoing intralysosomal decomposition might continue for a certain period to produce superoxide anion radicals (O2-) and thus also H2O2. This model of lipofuscinogenesis could satisfactorily explain the variations observed in the rates of lipofuscinogenesis among different postmitotic cell types in various species. Such variations might arise from a variety of factors including differences in the efficiency of the 'anti-oxidative shield', rate of H2O2 generation, amount of chain-breaking antioxidants, mode of intralysosomal iron chelation, rate of autophagocytosis as well as degree of efficiency of the intralysosomal hydrolytic enzymes.


Cell culture models for oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia.

According to the free radical theory of aging, loss of cellular function during aging is a consequence of accumulating subcellular damage inflicted by activated oxygen species. In cells, the deleterious effects of activated oxygen species may become manifest when the balance between radical formation and destruction (removal) is disturbed creating a situation denoted as 'oxidative stress'. Cell culture systems are especially useful to study the effects of oxidative stress, in terms of both toxicity and cellular adaptive responses. A better understanding of such processes may be pertinent to fully comprehend the cellular aging process. This article reviews three model systems for oxidative stress: extracellular sources of O2-. and H2O2, and normobaric hyperoxia (elevated ambient oxygen). Methodological and practical aspects of these exposure models are discussed, as well as their prominent effects as observed in cultures of Chinese hamster cell lines. Since chronic exposure models are to be preferred, it is argued that normobaric hyperoxia is a particularly relevant oxidative stress model for in vitro cellular aging studies.


Precise excision of transposons and point mutations induced by chemicals.

The ability of 23 chemicals (carcinogens and non-carcinogens) to induce precise excision of Tn10 and point mutations was studied in experiments with a single strain. The mutation assay was shown to detect a wider spectrum of genotoxic agents than the assay of Tn10 precise excision. The latter was induced only by potent SOS mutagens, which is in accordance with data on the SOS dependence of the induction of precise excision of Tn10. The precise excision assay as an additional test contributing to the knowledge of particular features of the action of a tested mutagen is discussed. The induction of precise excision of Tn10 by pyrene (and its failure to induce point mutations in this strain) demonstrates the value of using the transposon excision assay in cases of 'problem' mutagens.


The value of cytogenetic monitoring versus film dosimetry in the hot zone of a nuclear power plant.

Cytogenetic analysis was carried out in 41 workers prior to and following regular maintenance work in a nuclear power plant. Although film dosimetry did not show the maximal annual permitted dose in any of the examined subjects, cytogenetic analysis carried out following the work detected dicentric chromosomes in peripheral blood lymphocytes of 20 workers. According to our findings smoking habits and previous exposure to ionizing radiation had no effect on the increased number of chromosomal aberrations.


Proliferation index in bone marrow cells from severely malnourished rats during lactation.

The aim of this study was to determine if severe malnutrition affects the proportion of proliferating bone marrow cells in malnourished rats during lactation. Sister chromatid staining of metaphases was used as a parameter, as well as the morphology, size and color of bromodeoxyuridine labeled interphase nuclei. The BrdU proliferation labeling index was statistically lower in malnourished rats (20.4%), than in well-nourished controls (35.1%). A difference was also found between the two groups in the proportion of metaphases in first, second and third or successive cell cycle. The average generation time was longer in the malnourished group: 15.3 h, against 11.8 h for the controls. These results indicate that severe malnutrition affects both the proportion of bone marrow proliferating cells and their cell kinetics.


Interactive cytogenetic effects on rat bone-marrow due to chronic ingestion of 2,5,2',5' and 3,4,3',4' PCBs.

Rats who were fed low doses of single PCBs, either 2,5,2',5' or 3,4,3',4', did not demonstrate any chromosome breakage or mitotic changes in their bone marrow cells. However, there was a significant increase in chromosome damage observed in bone marrow cells of rats ingesting 10 ppm 2,5,2',5' plus 0.1 ppm 3,3,3',4' in combination. It is suggested that this PCB combination, previously found to cause superadditive chromosome damage in vitro, is also capable of causing chromosome damage in vivo, but these effects do not compromise cell proliferation because the mitotic index is not depressed.


Differences in sister-chromatid exchange frequency between homologous chromosomes in Muntiacus muntjak.

The frequency of sister-chromatid exchanges (SCEs) on homologous autosomes was studied in the peripheral blood lymphocytes of the Indian muntjac. The homologous autosomes differed from one another with respect to the SCE frequency on them.


Cytogenetic monitoring of farm animals under conditions of environmental pollution.

Cytogenetic examinations were carried out in 13 cattle farms, two herds of horses, one stag farm and 13 pig farms in areas with different levels of environmental contamination. The frequency of aberrant cells per 100 mitoses was 3.67 +/- 1.89 in pigs (n = 260) and 4.16 +/- 2.4 in herbivores (n = 497). This is a significant difference (p < 0.01). Ten times higher frequencies of chromatid exchanges were found in pigs. The examined herds were classified into three groups by the level of environmental contamination (satisfactory, impaired and severely impaired environment). Significant differences in aberrant cell counts were recorded between the groups of herbivorous animals. Significant differences in pigs were recorded only between herds in satisfactory and severely impaired environments. Significantly higher frequencies of aberrant cells were found in farms of herbivorous animals in the industrial area of Pardubice compared with findings in the South Moravian agricultural area (4.7% and 3.1% respectively). The effect of local contamination sources on farm environment was also investigated. A cattle farm located in the vicinity of a large chemical plant was examined five times at 6-month intervals. An autumn examination revealed significantly higher frequencies of aberrant cells compared with the spring examination.


Potentiation of sodium chromate(VI)-induced chromosomal aberrations and mutation by vitamin B2 in Chinese hamster V79 cells.

The effect of vitamin B2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 microM vitamin B2 (riboflavin) for 24 h prior to exposure to Na2CrO4 (2.5-5 microM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells.


Cytogenetic effects of sodium azide encapsulated in liposomes on heteroploid cell cultures.

Lipid vesicles (liposomes) have been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) we were able to deliver the classical mutagen, sodium azide, into human heteroploid HEp-2 cells. With this method sodium azide is not diluted in culture medium, but it is 'focused' into cells, producing chromosomal aberrations and other major genetic damages. Our results indicate that liposomes are suitable vectors for introducing clastogenic substances into cultured human cells.


Chromosome analysis in a population living in an area of Germany with the highest fallout deposition from the Chernobyl accident.

Chromosome analyses were carried out in 1989 in peripheral lymphocytes of 30 persons from the region in southeast Bavaria (Berchtesgaden) that received the highest radiocesium deposition (> 42 kBq/m2) in Germany. These persons belonged to a group for whom body burdens of radiocesium had been surveyed in 1987. Chromosome analyses had been performed by Stephan and Oestreicher for members of the same group in the period 1987-1990, and these authors have reported elevated rates of chromosome aberrations. Pohl-Rüling et al. (1991) have, likewise, concluded that there have been increased rates of chromosome aberrations in the population of the nearby Salzburg region in 1987. In the study reported here we do not see any elevations compared to our large pre-Chernobyl control group. The difference between our findings and the report of earlier increases cannot be fully explained in terms of the known temporal decrease of the rate of dicentrics and ring chromosomes.


Sleep deprivation in human males and its effect on SCE rates in chromosomes--a preliminary study.

The present preliminary study on 5 healthy volunteers deprived of sleep for a day showed a considerable increase in sister-chromatid exchange (SCE) rate in the chromosomes after sleep deprivation. Although the rate of increase varied between the studied subjects, interestingly the 5% plasma from a volunteer with a statistically significant increase after sleep deprivation, when supplemented to cell cultures from an age-matched normal subject, induced high SCEs. This suggests the presence/absence of some factor(s) in plasma samples of subjects showing high SCEs after sleep deprivation, playing a role in the induction of high SCEs in normal cells exposed to the samples in vitro. The present study also suggests that there could be categories which may or may not respond to sleep deprivation with high SCE induction and their plasma supplemented to normal cell culture in turn may or may not induce high SCEs. Nevertheless, the pathophysiological role of high SCEs induced by sleep deprivation in some cases remains intriguing and unresolved and needs further extensive study.


Sister-chromatid exchanges are increased in epileptics, but not by sodium valproate.

Sister-chromatid exchange (SCE) frequency has been studied from peripheral blood lymphocyte cultures of 21 patients with epilepsy on sodium valproate, 20 patients who had not started therapy (untreated) and 20 normal healthy controls. Treated and untreated patients with epilepsy were observed to have higher SCE frequencies (mean 9.05 and 9.82 respectively) than healthy controls (mean 4.8; P < 0.001). There was no significant difference in SCE frequency between treated and untreated patients. This suggests that the disease itself may be associated with an increased frequency of SCEs.


Spontaneous and induced levels of chromosomal aberration and sister-chromatid exchange in neurofibromatosis: no evidence of chromosomal hypersensitivity.

Chromosomal aberration (CA) and sister-chromatid exchange (SCE) frequencies have been assessed in 9 patients with von Recklinghausen's neurofibromatosis (NF1) and 8 apparently healthy controls. In separate experiments over a 5-year period, blood lymphocytes, skin fibroblast cell strains, and lymphoblastoid lines from both groups were treated with X-rays or mitomycin C (MMC) to determine whether the NF1 group was more sensitive to these agents than the control group. No difference between cells from NF1 patients and controls was observed with respect to spontaneous or X-ray-induced CA. Spontaneous or X-ray- and MMC-induced SCE frequencies were also similar in NF1 patients and controls.


Inducible protective processes in animal systems. III. Adaptive response of meiotic cells of the grasshopper, Poecilocerus pictus, to a low dose of ethyl methanesulfonate.

Meiotic cells of Poecilocerus pictus exposed to a low dose of 0.03 M of ethyl methanesulfonate (EMS) were found to be resistant to the induction of chromosomal anomalies by a subsequent challenge dose (0.12 M) of the same mutagen as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromosomal anomalies in metaphase I and II and anaphase I and II. These results indicate the presence of an inducible chromosomal repair mechanism in meiotic cells of P. pictus. The incidence of chromosome damage was found to be less when the time lag between the conditioning and challenging doses was reduced, suggesting that under the conditions tested, the efficacy of repair enzymes gradually decreases as the time between the two doses increases.


Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.


Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.


Detection of gamma-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka (Oryzias latipes) using AP-PCR fingerprinting.

Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of gamma-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which, to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F1 hybrids were the sum of those of their parents. Based on these findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy gamma-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy gamma-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of gamma-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.


An enhancement of the yield of X-ray-induced Minute mutations in the c3G female-ywmf-2 male system of Drosophila melanogaster.

The possible enhancement of the yield of X-ray-induced Minute mutations in the c3G female-ywmf-2 male system which is proposed to be responsible for the high production of spontaneous Minute mutations was investigated. To determine and compare the yield of X-ray-induced Minute mutations exactly, four series of crosses were made: (a) ywmf-2 male x Oregon-R (OrR) female crosses, spontaneous Minute mutations were scored; (b) ywmf-2 male x c3G female crosses, spontaneous Minute mutations produced in the c3G female-ywmf-2 male system were evaluated; (c) X-irradiated ywmf-2 male x OrR female crosses, the yield of Minutes induced by X-rays in the different stages of male germ cells was evaluated; and (d) X-irradiated ywmf-2 male x c3G female crosses, the yield of X-ray-induced Minutes in the c3G female-ywmf-2 male system was evaluated. The results show that the yield of X-ray-induced Minutes recorded in the c3G female-ywmf-2 male system is 2.37-16.55 times more than that in the ywmf-2 male x OrR female crosses. This finding clearly indicates that the yield of these mutations is greatly enhanced in the c3G female-ywmf-2 male system. This possibly suggests that the c3G female-ywmf-2 male system may be responsible not only for the high production of spontaneous Minute mutations but also for the high formation of radiation-induced Minutes.


Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.


Toxicity of some heavy metals in vivo and in vitro in Helianthus annuus.

To compare the toxicity of some heavy metals in vivo and in vitro, the effects of six metals, aluminum (Al), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) were studied in the oil-yielding plant Helianthus annuus. The percentage of seed germination and cytotoxic effects at different concentrations and durations of treatment as well as the growth rate of callus tissues in vitro were compared to ascertain the concentrations that can either support plant growth or cause lethality. Highest toxicity to the plant system was observed from the effects of Pb both at high and low concentrations whereas Zn was the least toxic; and similar effects were seen in vivo and in vitro. The clastogenic effects of Al, Cd, Cu, and Ni were dependent on concentration and length of treatment. Cu and Zn showed less severe cytotoxic damages than Al, Cd, Pb, and Ni. In vitro growth could be supported at 100-1000 times the diluted concentrations of the metals in comparison to in vivo treatment.


Detection of oxidative mutagens in an urban air-particulate extract: a preliminary study.

The Ames assays strains TA98 and TA100 have been useful in characterizing complex mixtures from organic solvent extracts of particles from diesel-powered vehicles, ambient air, and other sources. In this paper we report preliminary experiments using TA102, a bacterial strain that detects compounds that can oxidize DNA, to characterize the mutagenicity of an ambient air sample collected in Ann Arbor, MI. Four sets of ambient air filters were collected in duplicate over a period of several days. The mutagenicities of methylene chloride extracts of these filters were compared using strains TA98, TA100 and TA102. The concentration-mutagenicity data for TA98 and TA100 were linear over the concentration range 0-200 micrograms extract/plate. The mutagenicity of the extracts using TA102 was much lower than the other two strains and was non-linear over the concentration range tested. These results suggest that it would be difficult to use TA102 to identify the oxidative mutagens present in an ambient air particulate extract.


The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.


Neither enhanced removal of cyclobutane pyrimidine dimers nor strand-specific repair is found after transcription induction of the beta 3-tubulin gene in a Drosophila embryonic cell line Kc.

Nucleotide excision repair (NER) of ultraviolet (UV) light induced cyclobutane pyrimidine dimers (CPDs) was assayed in a Drosophila melanogaster Kc subline that responds to treatment with the steroid hormone 20-hydroxyecdysone (20-OH-E; beta-ecdysone, ecdysterone). In this cell line the hormone induces transcription of the beta 3-tubulin gene which is not expressed under standard culture conditions. Cells were exposed to either 10 or 15 J/m2 UV (predominantly 254-nm) and removal of CPDs from several genes, including beta 3-tubulin, and total cellular DNA was assayed. We show that upon induction of transcription of the beta 3-tubulin gene, its repair is not enhanced. In non-treated as well as 20-OH-E treated cells, repair kinetics in beta 3-tubulin resemble those in the active genes Gart and Notch, the inactive locus white and total cellular DNA. Moreover, in the presence as well as in the absence of transcription, the separate strands of the beta 3-tubulin gene are repaired with the same rate and to the same extent: about 90% after 24 h. It can be concluded from these observations that transcription is not a prerequisite for the efficient repair of CPDs in the Drosophila embryonic Kc cell line.


Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.


Suppression of X-ray-induced chromosome aberrations in ataxia telangiectasia cells by introduction of a normal human chromosome 11.

We studied X-ray-induced chromosome aberrations in ataxia telangiectasia (AT) cells containing an introduced chromosome 11 or 12 derived from normal human fibroblasts. We used microcell-mediated chromosome transfer to introduce the normal chromosomes into AT cells belonging to complementation group D. Cells were irradiated with 1 Gy of X-rays in the G2 phase. All 5 hybrid clones with an introduced chromosome 11 showed a reduction in the frequency of chromatid-type aberrations to normal levels, whereas all 4 hybrid clones with an introduced chromosome 12 failed to show this reduction. This finding, taken together with our previous report that chromosome 11 can restore radioresistant cell killing in AT cells, indicates that a defective gene on chromosome 11 in AT cells is responsible for the hypersensitivity to not only cell killing but also chromosome aberrations. Our results suggest that a putative AT gene on chromosome 11 plays an important role in the repair process of radiation-induced DNA damage that leads to chromosome aberrations.


Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.


Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.


A re-examination of the intragenome distribution of repaired sites in proliferating xeroderma pigmentosum complementation group C fibroblasts.

We find that rapidly proliferating fibroblasts from xeroderma pigmentosum complementation group C (XP-C) patients, cells that have a small residual DNA excision repair capacity, repair DNA in localized regions of the genome in a clustered pattern rather than at single sites in dispersed locations. This finding is similar to that observed earlier for nondividing cells but is in contrast to published results that indicate that the residual repair in proliferating XP-C cells is dispersed throughout the genome in a non-clustered pattern. While we detect the same amount of repair in both proliferating and nondividing cells, we also observe no shift from the clustered pattern of repair to a more dispersive pattern when nondividing cells are stimulated to proliferate by fresh serum addition. We have no obvious explanation for these discrepancies with the published results. We have noted previously that proliferating XP-C cells are very UV sensitive relative to normal cells while nondividing cells that exhibit the same amount of repair activity are relatively UV resistant. There is no satisfactory explanation for this change in relative response to the lethal effects of UV, a change not observed for cell strains from other XP complementation groups. However, we argue that clustered repair in specific genomic regions promotes survival in nondividing XP-C cells but does not promote survival in proliferating cells.


New DNA repair-deficient mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas reinhardtii, uvs12 and uvs13, were characterized. Genetic analysis proved that they were non-allelic. They complemented the other repair-deficient mutants uvs8, uvs9, uvs10 and uvs11. While uvs12 may have an impaired excision-repair pathway, uvs13 is, by its UV sensitivity under non-photoreactivating conditions, very similar to uvsE1 and uvs10, but differs in the effect of caffeine on its survival. After UV, survival of some repair-deficient mutants was, under photoreactivating conditions, much lower than that of phr1, while survival of other repair-deficient mutants did not differ from that of a wild-type strain. A lower UV survival of some dark-repair-defective mutants of Chlamydomonas reinhardtii, under photoreactivating conditions, can perhaps be used as an additional criterion for mutants defective in the excision-repair pathway.


Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.


Recent advances in DNA repair and mutation: a report of the meeting of the British Photobiology Society and the DNA Repair Network.

The 1991 joint meeting of the British Photobiology Society and the DNA Repair Network was held in London on the 16th of December at the Polytechnic of East London. Despite the best efforts of the IRA to disrupt transport in London, a full programme of eighteen talks were presented on the theme of "Recent Advances in DNA Repair and Mutation".


Inorganic mercury is transported from muscular nerve terminals to spinal and brainstem motoneurons.

The distribution of mercury within the brainstem and spinal cord of mice was investigated with the autometallographic technique after intramuscular administration of a single dose of mercuric mercury (HgCl2). Deposits of mercury were localized to motor neurons of the spinal cord and to brainstem motor nuclei; i.e., neurons with their peripheral projections outside the blood-brain barrier. Unilateral ligation of the hypoglossal nerve prior to the injection of HgCl2 prevented the accumulation of mercury deposits in the ipsilateral hypoglossal nucleus. The selective accumulation of mercury in spinal and brainstem motoneurons is most probably due to a leakage of metal-protein complexes from capillaries in muscle into myoneural junctions, followed by uptake into nerve terminals and retrograde axonal transport. The possible link between this process and the development of motor neuron degeneration in ALS is discussed.


The effect of finasteride in men with benign prostatic hyperplasia. The Finasteride Study Group.

Benign prostatic hyperplasia is a progressive, androgen-dependent disease resulting in enlargement of the prostate gland and urinary obstruction. Preventing the conversion of testosterone to its tissue-active form, dihydrotestosterone, by inhibiting the enzyme 5 alpha-reductase could decrease the action of androgens in their target tissues; in the prostate the result might be a decrease in prostatic hyperplasia and therefore in symptoms of urinary obstruction. In a double-blind study, we evaluated the effect of two doses of finasteride (1 mg and 5 mg) and placebo, each given once daily for 12 months, in 895 men with prostatic hyperplasia. Urinary symptoms, urinary flow, prostatic volume, and serum concentrations of dihydrotestosterone and prostate-specific antigen were determined periodically during the treatment period. As compared with the men in the placebo group, the men treated with 5 mg of finasteride per day had a significant decrease in total urinary-symptom scores (P less than 0.001), an increase of 1.6 ml per second (22 percent, P less than 0.001) in the maximal urinary-flow rate, and a 19 percent decrease in prostatic volume (P less than 0.001). The men treated with 1 mg of finasteride per day did not have a significant decrease in total urinary-symptom scores, but had an increase of 1.4 ml per second (23 percent) in the maximal urinary-flow rate, and an 18 percent decrease in prostatic volume. The men given placebo had no changes in total urinary-symptom scores, an increase of 0.2 ml per second (8 percent) in the maximal urinary-flow rate, and a 3 percent decrease in prostatic volume. The frequency of adverse effects in the three groups was similar, except for a higher incidence of decreased libido, impotence, and ejaculatory disorders in the finasteride-treated groups. The treatment of benign prostatic hyperplasia with 5 mg of finasteride per day results in a significant decrease in symptoms of obstruction, an increase in urinary flow, and a decrease in prostatic volume, but at a slightly increased risk of sexual dysfunction.


Environmental exposure to lead and children's intelligence at the age of seven years. The Port Pirie Cohort Study.

Exposure to lead in early childhood is thought to result in delayed neuropsychological development. As yet there is little longitudinal evidence to establish whether these effects persist into later childhood. We measured IQ scores in 494 seven-year-old children from the lead-smelting community of Port Pirie, Australia, in whom developmental deficits associated with elevated blood lead concentrations had already been reported at the ages of two and four years. Exposure to lead was estimated from the lead concentrations in maternal blood samples drawn antenatally and at delivery and from blood samples drawn from the children at birth (umbilical-cord blood), at the ages of 6 and 15 months and 2 years, and annually thereafter. Data relating to known covariates of child development were collected systematically for each child throughout the first seven years of life. We found inverse relations between IQ at the age of seven years and both antenatal and postnatal blood lead concentrations. After adjustment by multiple regression for sex, parents' level of education, maternal age at delivery, parents' smoking status, socioeconomic status, quality of the home environment, maternal IQ, birth weight, birth order, feeding method (breast, bottle, or both), duration of breast-feeding, and whether the child's natural parents were living together, the relation with lead exposure was still evident for postnatal blood samples, particularly within the age range of 15 months to 4 years. For an increase in blood lead concentration from 10 micrograms per deciliter (0.48 mumol per liter) to 30 micrograms per deciliter (1.45 mumol per liter), expressed as the average of the concentrations at 15 months and 2, 3, and 4 years, the estimated reduction in the IQ of the children was in the range of 4.4 points (95 percent confidence interval, 2.2 to 6.6) to 5.3 points (95 percent confidence interval, 2.8 to 7.8). This reduction represents an approximate deficit in IQ of 4 to 5 percent. Low-level exposure to lead during early childhood is inversely associated with neuropsychological development through the first seven years of life.


Comparison of a second-generation combination chemotherapeutic regimen (m-BACOD) with a standard regimen (CHOP) for advanced diffuse non-Hodgkin's lymphoma.

In 1984, the Eastern Cooperative Oncology Group began a randomized controlled clinical trial of patients with advanced (stage III or IV) diffuse mixed or diffuse large-cell lymphoma to determine whether complete-remission rates, survival, and toxicity differed when patients were treated with a chemotherapeutic regimen containing cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), as compared with a regimen containing bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, methotrexate, and leucovorin (m-BACOD). From July 1984 through January 1988, 392 patients were enrolled, 325 of whom (83 percent) were eligible for the analysis and capable of being evaluated. The extent of disease was defined according to standard staging techniques, including bilateral bone-core biopsies in 88 percent of patients. Randomization was stratified according to age (< 60 or > or = 60 years), performance status (0, 1, or other), stage (III or IV), and histologic presentation (diffuse mixed or diffuse large-cell lymphoma). After a median follow-up of four years, there were no significant differences in rates of complete remission, time to treatment failure, disease-free survival, or overall survival in the patients treated with CHOP as compared with those treated with m-BACOD. However, there was more severe and life-threatening pulmonary, infectious, and hematologic toxicity associated with the m-BACOD regimen. In an attempt to measure the importance of dose intensity in the 325 patients who could be analyzed, we retrospectively calculated dose intensity (measured in milligrams per square meter of body-surface area per week) and normalized dose intensity (defined as a percentage of the prescribed dose) for all drugs. The median normalized dose intensity for both cyclophosphamide and doxorubicin was found to be greater in the patients treated with CHOP than in those treated with m-BACOD. For patients with stage III or IV diffuse mixed or diffuse large-cell lymphoma, CHOP is superior to m-BACOD, but the role of dose intensity is not yet clear.


Chemotherapy of advanced Hodgkin's disease with MOPP, ABVD, or MOPP alternating with ABVD.

MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) has been the standard treatment for Hodgkin's disease for almost 20 years. In a randomized, multicenter trial, we compared three regimens of primary systemic therapy for newly diagnosed advanced Hodgkin's disease in Stages IIIA2, IIIB, and IVA or IVB: (1) MOPP alone given for 6 to 8 cycles, (2) MOPP alternating with ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) for 12 cycles, and (3) ABVD alone for 6 to 8 cycles. Patients in a first relapse after radiation therapy were eligible. No additional radiation therapy was given. Patients who did not have a complete response or who had a relapse with either MOPP alone or ABVD alone were switched to the opposite regimen. Of 361 eligible patients, 123 received MOPP, 123 received MOPP alternating with ABVD, and 115 received ABVD alone. The patients were stratified according to age, stage, previous radiation, histologic features, and performance status. The overall response rate was 93 percent, with complete responses in 77 percent: 67 percent in the MOPP group, 82 percent in the ABVD group, and 83 percent in the MOPP-ABVD group (P = 0.006 for the comparison of MOPP with the other two regimens, both of which contained doxorubicin). The rates of failure-free survival at five years were 50 percent for MOPP, 61 percent for ABVD, and 65 percent for MOPP-ABVD. Age, stage (III vs. IV), and regimen influenced failure-free survival significantly. Overall survival at five years was 66 percent for MOPP, 73 percent for ABVD, and 75 percent for MOPP-ABVD (P = 0.28 for the comparison of MOPP with the doxorubicin regimens). MOPP had more severe toxic effects on bone marrow than ABVD and was associated with greater reductions in the prescribed dose. In this trial, ABVD therapy for 6 to 8 months was as effective as 12 months of MOPP alternating with ABVD, and both were superior to MOPP alone in the treatment of advanced Hodgkin's disease. ABVD was less myelotoxic than MOPP or ABVD alternating with MOPP.


A role for hepatitis C virus infection in type II cryoglobulinemia.

Type II cryoglobulinemia is a vasculitis characterized by cryoglobulins consisting of complexes of polyclonal IgG and monoclonal IgM rheumatoid factors. The cause of these immune complexes is unknown, though both the hepatitis B (HBV) and C (HCV) viruses have been suspected. We studied 19 patients with Type II cryoglobulinemia for markers of HCV and HBV infection. Quantitative HCV antibody and RNA studies were performed on whole serum, cryoprecipitates, and supernatants. Eight patients (42 percent) had HCV antibodies, and 16 (84 percent) had HCV RNA: Of the 19 patients, 5 (26 percent) had HBV markers, but only 1 had evidence of active HBV infection. Control serum samples from nine patients with Type I cryoglobulinemia were negative for HCV antibody and HCV RNA: There was a close, although not exclusive, association of one type of rheumatoid factor (WA) with HCV RNA: HCV antibody and HCV RNA were concentrated approximately 10-fold and 1000-fold, respectively, in the Type II cryoglobulins examined. Type II cryoglobulinemia is strongly associated with concomitant HCV infection and a high rate of false negative serologic tests. HCV virions and HCV antigen-antibody complexes are concentrated in the cryoprecipitates, most commonly in association with the WA type of rheumatoid factor, suggesting a role for HCV in the pathogenesis of mixed cryoglobulinemia.


Effect of keratin on the lipid composition of Scopulariopsis brevicaulis (Bainier).

Lipid content and composition of Scopulariopsis brevicaulis cultured with or without its natural substrate keratin was investigated. Lipid content was found to be lower in the presence of keratin (maximum 12% against 19% in the absence of keratin), with differences in levels of linoleic acid (C18 = 2), palmitic (C16:0) and to a lesser extent palmitoleic (C16 = 1) and alpha-linolenic (C18 = 3) acids. The degree of unsaturation was correspondingly lower in the organisms cultured with keratin as substrate.


Development of buprenorphine for the treatment of opioid dependence.

Data from these studies indicate that buprenorphine is efficacious in treating opioid dependence. It was possible to induct heroin addicts rapidly onto buprenorphine without precipitating an opioid withdrawal syndrome. A daily 8-mg SL dosage was sufficient to maintain individuals without producing reports of withdrawal symptoms. When buprenorphine was administered at the above dose every other day, however, mild withdrawal symptoms were reported, and responses to challenges with intravenously given hydromorphone appeared greater than when the challenges were given intramuscularly. From these results, the authors conclude that buprenorphine at this dose should be administered on a daily basis. These results are now being applied to a phase II outpatient clinical trial comparing buprenorphine with methadone.


Amyloid beta-peptide is produced by cultured cells during normal metabolism.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.


Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.


Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase.

The kinase activity of pp60c-src is specifically and transiently increased during mitosis and repressed during interphase. Loss of cell-cycle control of pp60c-src occurs on mutation of Tyr527 to Phe or when pp60c-src is associated with polyoma middle-T-antigen, and these conditions result in cell transformation or tumorigenesis. In both cases, pp60c-src has elevated kinase activity which is maintained throughout the cell cycle and accompanied by dephosphorylation of the carboxy-terminal negative regulatory Tyr527 site, or mimicry of Tyr527 dephosphorylation in the case of the mutant. Here we report that overexpression of the receptor-like protein tyrosine phosphatase PTP alpha results in persistent activation of pp60c-src kinase, with concomitant cell transformation and tumorigenesis. In PTP alpha-overexpressing cells, the pp60c-src kinase activation is accompanied by dephosphorylation at Tyr527, and direct dephosphorylation of this site by purified PTP alpha occurs in vitro. Our results suggest that PTP alpha is involved in the regulation of cell proliferation, exerting at least some of its effects through pp60c-src kinase, and has oncogenic capability when overexpressed.


Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic.

Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity.