Estimation of haemopoietic progenitor cells in peripheral blood by the Advia 120 and BD vantage flow cytometer: a direct comparison for the prediction of adequate collections.

Peripheral blood stem cells are increasingly used to ensure rapid haematological engraftment after myeloablative chemotherapy. After mobilization, progenitor cells in the blood can be enumerated to predict an adequate collection by leukapheresis. The Advia 120 automated counter has an immature cell channel measuring a parameter known as large undifferentiated cells (LUC's), which were quantified to assess their value in refining the timing of apheresis. Data were available from 102 apheresis sessions. Positive correlation was found for peripheral blood CD34+ cells and apheresis counts (r = 0.82, P < 0.0005) but not for total WCC (r = -0.15, P = 0.13) or LUC count (r = 0.12, P = 0.23). Our results indicate that the LUC population in peripheral blood has no relevance to the subsequent CD34 content of the apheresis product and CD34 cell enumeration by flow cytometry is advocated.

Reduction and Mutagenic Activation of Nitroaromatic Compounds by a Mycobacterium sp.

Volume 60, no. 12, p. 4264: Figure 1B should appear as shown below. [This corrects the article on p. 4263 in vol. 60.].

Reduction and mutagenic activation of nitroaromatic compounds by a Mycobacterium sp.

Mycobacterium sp. strain Pyr-1 cells, which were grown to the stationary phase in media with and without pyrene, were centrifuged and resuspended in a medium containing 1-nitropyrene. Cells that had been grown with pyrene oxidized up to 20% of the added 1-nitropyrene to 1-nitropyrene-cis-9,10- and 4,5-dihydrodiols. However, cells that had been grown without pyrene reduced up to 70% of the 1-nitropyrene to 1-aminopyrene but did not produce dihydrodiols. The nitroreductase activity was oxygen insensitive, intracellular, and inducible by nitro compounds. Nitroreductase activity was inhibited by p-chlorobenzoic acid, o-iodosobenzoic acid, menadione, dicumarol, and antimycin A. Extracts from cells that had been grown without pyrene activated 1-nitropyrene, 1-amino-7-nitrofluorene, 2,7-dinitro-9-fluorenone, 1,3-dinitropyrene, 1,6-dinitropyrene, and 6-nitrochrysene to DNA-damaging products, as shown in Salmonella typhimurium tester strains by the reversion assay and by induction of the umuC gene. Activation of nitro compounds, as shown by the umu test, was enhanced by NADPH. This study shows that Mycobacterium sp. strain Pyr-1 metabolizes nitroaromatic compounds by both oxidative and reductive pathways. During reduction, it generates products that are mutagenic.

Molecular analysis of DNA adducts and hprt mutations produced by 6-nitrosochrysene in Chinese hamster ovary cells.

We have characterized the mutational spectrum of 6-nitrosochrysene in the hprt gene of Chinese hamster ovary (CHO-K1) cells and also examined the adducts formed by this compound in CHO-K1 cells by quantitative 32P-postlabeling analysis. Seventy percent of the identified mutations were simple basepair substitutions, and they occurred more often at A:T (14/17) than at G:C. Furthermore, 13 of the basepair substitutions at A:T had the mutated dA, the probable adducted residue, on the non-transcribed DNA strand. The preference for mutation at A:T contrasted sharply with the distribution of adducts formed by 6-nitrosochrysene: 80% of the identified adducts were with dG, while only 20% were probably formed through binding with dA. Analyses conducted with excision-repair-defective CHO-UV5 cells revealed both a preference for basepair substitution at A:T and an adduct profile that were similar to those found for repair-proficient CHO-K1 cells. However, basepair substitutions from CHO-UV5 cell mutants had the mutated dAs distributed randomly between the non-transcribed and transcribed DNA strands. The mutational spectra found for solvent control CHO-K1 and CHO-UV5 cells differed from those of the 6-nitrosochrysene-treated cultures. These findings suggest that 6-nitrosochrysene-induced mutations are targeted to DNA damage, but that 6-nitrosochrysene-derived dA adducts are much more effective at producing mutations than 6-nitrosochrysene-derived dG adducts. The extreme strand bias for mutated dAs in the CHO-K1 mutational spectrum appears to result from preferential removal of 6-nitrosochrysene-induced DNA lesions from the transcribed DNA strand.

32P-postlabelling in studies of arylamine and nitroaromatic hydrocarbon activation and mutagenesis.

Carcinogenic arylamines and nitroaromatic hydrocarbons are chemicals that present occupational health hazards and share pathways of metabolic activation. The 32P-postlabelled DNA adducts formed in Chinese hamster ovary (CHO) cells treated with metabolites from two pathways that are common to the activation of the nitroaromatic hydrocarbon 6-nitrochrysene (6-NC) and the arylamine 6-aminochrysene (6-AC) compared with the spectra of mutations induced at the CHO hprt locus by these were metabolites. 6-Nitrosochrysene (6-NOC), which is reduced by the cells to N-hydroxy-6-AC, formed adducts mainly with deoxyguanosine, but induced mutations primarily through base-pair substitution involving deoxyadenosine. In contrast, 6-AC 1,2-dihydrodiol produced DNA adducts and mutations that mainly involved deoxyguanosine residues. The two major activation pathways for 6-NC and 6-AC thus produce distinct adduct and mutation spectra in CHO cells, and these adduct and mutational spectra are different from those of other arylamines and nitroaromatic hydrocarbons that have been studied.

DNA sequence analysis of 1-nitrosopyrene-induced mutations in the hprt gene of Chinese hamster ovary cells.

DNA sequence was determined in 21 mutants induced at the hprt locus of Chinese hamster ovary (CHO) cells by 1-nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene. Following cDNA synthesis using RNA from each of the mutants, the hprt protein-coding region was amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequence analysis. Sixteen primary mutations were found: seven were G:C----T:A transversions, five were G:C----A:T transitions, two were single basepair insertions, one was a single basepair deletion, and one was a complex mutation involving substitutions at two A:T basepairs. The simple basepair substitution mutations preferentially occurred with one or two purines 3' to the mutated dG, and mutations in exons 1-4 disproportionately occurred with the mutated dG on the nontranscribed DNA strand. In addition, 12 of the mutants produced one or more cDNA PCR products with partial or complete exon deletions. Seven mutants with multiple PCR products had point mutations in one of the products; exon deletions in the other product(s) removed these point mutations. A group of solvent control mutants had a different distribution of basepair substitution mutations and a lower proportion of cDNAs with exon deletions than that found for the 1-nitrosopyrene-induced mutants. The results indicate a specificity for the induction of mutations in the hprt gene of CHO cells by 1-nitrosopyrene with respect to both the types of mutations produced and their location in the hprt gene. Also, the elimination of point mutations in many of the cDNA PCR products with exon deletions suggests that mutations in the protein-coding sequence affect hprt mRNA processing.

Analysis of solvent control and 1-nitrosopyrene-induced Chinese hamster ovary cell mutants by Southern and northern blots and the polymerase chain reaction.

1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). Pstl- and BamHl-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequence. These alterations may contribute to the 6-thioguanine-resistant phenotype.

Relationship between life expectancy and endogenous DNA single-strand breakage, strand break induction and DNA repair capacity in the adult housefly, Musca domestica.

The objective of this study was to investigate the relationship between genomic damage and the physiological rate of aging. Endogenous DNA single-strand breaks, susceptibility of DNA to exogenously induced strand breaks and the capacity to repair strand breakage were compared, using the alkaline elution technique, in flies of the same chronological age but with different life expectancy. Distinctions between physiological and chronological ages were made (1) by experimentally altering the life spans of houseflies by varying the level of physical activity, and (2) by phenotypic selection of short- and long-lived cohorts from the same population. The degree of endogenous DNA single-strand breaks was found to be unrelated to physiological age. However, flies selected for relatively shorter life expectancy exhibited a greater susceptibility to exogenously-induced (gamma-irradiation) single-strand breakage. Flies with a longer life expectancy exhibited a more efficient repair capacity to reverse single-strand breakage than those with a shorter life expectancy.

Effect of age on endogenous DNA single-strand breakage, strand break induction and repair in the adult housefly, Musca domestica.

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular senescence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by gamma-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results do not support the view that DNA single-strand breaks are a causal factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation.

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum.

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.

Iron induces oxidative stress and may alter the rate of aging in the housefly, Musca domestica.

Iron is known to play a catalytic role in the generation of oxygen free radicals in vitro. The present study was conducted in order to determine the in vivo effects of iron intake. Administration of 2 mM ferrous chloride to adult male houseflies in their drinking water significantly shortened their life span, increased the concentration of inorganic peroxides and chloroform-soluble fluorescent material, and stimulated the activity of catalase. Levels of superoxide dismutase activity, glutathione and oxygen utilization were unaffected. Overall, these results indicate that iron causes oxidative stress in vivo and may influence the rate of aging.

Effects of exogenous antioxidants on the levels of endogenous antioxidants, lipid-soluble fluorescent material and life span in the housefly, Musca domestica.

Effects of exogenous antioxidant administration (0.5% and 2% ascorbate, beta-carotene and alpha-tocopherol in sucrose) on life-span, metabolic rate, activities of superoxide dismutase and catalase, levels of glutathione, inorganic peroxides and chloroform-soluble fluorescent material (lipofuscin) were examined in adult male houseflies. Administration of antioxidants at a level of 0.5% did not affect life-span, whereas, 2% ascorbate and alpha-tocopherol decreased average life-span. Metabolic rate of flies was unaffected, except by 2% ascorbate, which caused a decrease. Superoxide dismutase activity was depressed by 2% ascorbate at all ages, and by beta-carotene and alpha-tocopherol in older flies. Catalase activity was unaffected except by alpha-tocopherol at younger ages. Glutathione concentration was decreased by ascorbate and beta-carotene at both concentrations administered. Inorganic peroxides (H2O2) were increased by 2% beta-carotene and alpha-tocopherol. Only high concentrations of ascorbate and beta-carotene decreased the level of soluble fluorescent material. Results suggest that administration of exogenous antioxidants causes a compensatory depression of endogenous defenses.

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica.

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum: (cellular differentiation/Physarum/oxy-free radicals/superoxide dismutase/glutathione).

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.

Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly.

The effects of oxidative stress in the adult male housefly were examined by the administration of 1 mM paraquat. Houseflies exhibit NADH and NADPH-diaphorase activity. Paraquat caused a significant decrease in life span, metabolic rate and the concentration of thiobarbituric acid-reactants. Concentrations of reduced glutathione and inorganic peroxides were increased by paraquat. Paraquat stimulated the activity of catalase but did not affect activities of superoxide dismutase and glutathione reductase. The levels of oxidized glutathione and the rate of fluorescent age pigment accumulation were unaffected by paraquat. Results indicate that paraquat toxicity does not result from lipid peroxidation.

Abnormal peripheral blood lymphocytes and bone marrow infiltration in non-Hodgkin's lymphoma.

The blood lymphocytes of 21 normal blood specimens, 20 patients with various malignant disorders and 63 patients with non-Hodgkin's disease lymphoma, all with lymphocyte counts below 6 x 10(9)/l, were examined by sheep red cell rosetting, fluorescent antisera to surface immunoglobulin and for sensitivity to colchicine. The results were correlated with bone marrow infiltration and lymph node histological findings. The ratio of kappa to lambda light chains in the surface immunglobulin was used to determine if an abnormal clone of lymphocytes was present. In the normals and the non lymphoma controls the expected normal ratio of approximately 2K to 1 lambda was found with a narrow spread. In non-Hodgkin's lymphoma cases there was a wide spread of ratios with half the cases outside the range of the controls. These cases were considered to have a clone of abnormal lymphocytes in the blood although their routine blood and differential counts were usually normal. There was a very significant correlation of the presence of a clone with ultrasensitivity to colchicine and with involvement of the bone marrow. 81% of the cases with a histological diagnosis of well-differentiated diffuse lymphocytic lymphoma, 64% of those with poorly differentiated diffuse but only 22% of those with diffuse histiocytic had an abnormal lymphocyte clone demonstrated by an abnormal K:lambda ratio.