A novel 96-well scintillation proximity assay for the measurement of apoptosis.

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca(2+), the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T(R)). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[(35)S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.

DNA sequence analysis of methylene chloride-induced HPRT mutations in Chinese hamster ovary cells: comparison with the mutation spectrum obtained for 1,2-dibromoethane and formaldehyde.

Glutathione-S-transferase-mediated metabolism of methylene chloride (MC) generates S-chloromethylglutathione, which has the potential to react with DNA, and formaldehyde, which is a known mutagen. MC-induced mutations in the HPRT gene of Chinese hamster ovary cells have been sequenced and compared with the mutations induced by 1, 2-dibromoethane (1,2-DEB), which is known to act through a glutathione conjugate, and formaldehyde. All three compounds induced primarily point mutations, with a small number of insertion and deletion events. The most common point mutations induced by MC were GC-->AT transitions (4/8), with two GC-->CG transversions and two AT-->TA transversions. This pattern of mutations showed greater similarity with 1,2-DBE, where the dominant point mutations were GC-->AT transitions (7/9), than formaldehyde, where all mutations were single base transversions and 5/6 occurred from AT base pairs. The mutation sequence results for MC suggest that S-chloromethylglutathione plays a major role in MC mutagenesis, with only a limited contribution from formaldehyde. The involvement of a glutathione (GSH) conjugate in MC mutagenicity would be analogous to the well-characterized pathway of activation of 1,2-DBE.

Mouse liver glutathione S-transferase mediated metabolism of methylene chloride to a mutagen in the CHO/HPRT assay.

Although methylene chloride (MC) is readily detectable as a bacterial mutagen, published studies in mammalian cells have been inconclusive. We have previously shown (Graves et al., 1995) that glutathione S-transferase (GST)-mediated metabolism of MC by mouse liver cytosol (S100 fraction) causes DNA single-strand (ss) breaks in CHO cells. In this study, MC GST metabolites were shown to cause mutations at the HPRT locus of CHO cells. The mutagenicity of MC was enhanced by exposing the cells in suspension rather than as attached cultures. The MC GST metabolite formaldehyde was mutagenic in independent experiments, although the number of mutants induced was lower than with the MC. CHO HPRT mutations were also induced by the reference genotoxin 1,2-dibromoethane (1,2-DBE), which is activated to a mutagen by GST-mediated metabolism. Assay of DNA ss breaks and DNA-protein cross-links at mutagenic concentrations of MC, formaldehyde or 1,2-DBE, showed that all three compounds induced DNA ss breaks, but only formaldehyde induced significant DNA-protein cross-linking. These results suggest that whilst formaldehyde may play a role in MC mutagenesis, its weak mutagenicity and the absence of significant DNA-protein cross-linking after MC exposure, leads to the conclusion that the MC DNA damage and resulting mutations are induced by the glutathione conjugate of MC, S-chloromethylglutathione.

Methylene chloride-induced DNA damage: an interspecies comparison.

DNA single-strand (ss) breaks were detected in the livers of B6C3F1 mice immediately following exposure to 4000-8000 p.p.m. methylene chloride (MC) for 6 h. This damage was undetectable 2 h after exposure, suggesting an active DNA repair process. Similarly, DNA ss breaks were detected in whole lung homogenates taken from mice exposed to 2000-6000 p.p.m. MC. The DNA of mouse Clara cells incubated in vitro with MC was also damaged at concentrations of 5 mM MC and above. Pre-treatment of mice with the glutathione depletor buthionine sulphoximine (BSO) caused a decrease in the amount of DNA damage detected, suggesting a GST-mediated mechanism. DNA damage was also reduced in Clara cells when incubated in vitro with MC in the presence of BSO. In CHO cells induction of DNA damage was dependent upon exogenous MC metabolism by mouse liver S100 fraction (but not microsomes) in the presence of GSH. DNA ss breaks were not induced by MC in hamster hepatocytes in vitro at concentrations from 5 to 90 mM MC, nor in eight individual samples of normal human hepatocytes exposed to MC at similar concentrations. The ability of MC to induce DNA ss breaks in the four species studied is entirely compatible with the known carcinogenicity of this chemical in animals and offers experimental evidence to suggest that humans would not be susceptible to MC-induced liver cancer. The DNA ss breaks correlate with the metabolism of MC by the GST pathway and provide an explanation for the lack of sensitivity of hamsters and rats to MC-induced liver cancer.

Relations between upper limb soft tissue disorders and repetitive movements at work.

To make a preliminary assessment of whether upper limb soft tissue disorders might be associated with activities at work, we have conducted a case-control study of subjects attending orthopedic clinics in three cities. All subjects between the ages of 16 and 65 years, in whom defined soft tissue conditions of the upper limb were diagnosed by the participating orthopedic surgeons, were invited to take part. Controls were subjects attending the same clinics within the same age range whose clinical diagnosis did not include disease of the upper limb, cervical or thoracic spine. Information concerning repetitive movements of the upper limbs at work was elicited by questionnaire. Five hundred eighty cases and 996 controls were studied, representing 96% and 93%, respectively, of those invited to participate. The diagnoses of the cases included soft tissue conditions affecting the shoulder, elbow, forearm, wrist, thumb, hand, and fingers. The diagnoses of the controls included traumatic, degenerative, and inflammatory conditions, mostly of the legs and lower back. Women predominated among the cases (70%) and men among the controls (56%). Of 221 female cases with injury to the wrist and forearm, 32 were cleaner/domestics (14.5%) compared to 35 to 439 controls (8%), a difference statistically significant at the 2 1/2% level. Other jobs significantly overrepresented (5% level) among female cases with injuries at various anatomical sites included hairdressers, secretary/temps, assembly line workers, and machine operators (type unspecified). Among male cases, electricians were significantly overrepresented (5% level). Jobs for which there was a suggestion (p < 0.1) of overrepresentation among cases included butchers and teacher/lecturers (both males only) and the combined job groups (chosen a priori for analysis) of keyboard operators, machine operators, and music teachers (all three jobs, females only).

Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.

The role of formaldehyde and S-chloromethylglutathione in the bacterial mutagenicity of methylene chloride.

Methylene chloride was less mutagenic in Salmonella typhimurium TA100/NG-11 (glutathione-deficient) compared to TA100, indicating that glutathione is involved in the activation of methylene chloride to a mutagen in bacteria. In rodents, the pathway of methylene chloride metabolism utilizing glutathione produces formaldehyde via a postulated S-chloromethylglutathione conjugate (GSCH2Cl). Formaldehyde is known to cause DNA-protein cross-links, and GSCH2Cl may act as a monofunctional DNA alkylator by analogy with the glutathione conjugates of 1,2-dihaloalkanes. The lack of sensitivity of Salmonella TA100 towards formaldehyde (Schmid et al., Mutagenesis, 1 (1986) No. 6, 427-431) suggests that GSCH2Cl is responsible for methylene chloride mutagenicity in Salmonella. In Escherichia coli K12 (AB1157), formaldehyde was mutagenic only in the wild-type, a characteristic shared with cross-linking agents, whereas 1,2-dibromoethane (1,2-DBE) was more mutagenic in uvrA cells (AB1886). Methylene chloride, activated by S9 from mouse liver, was mutagenic only in wild-type cells, suggesting a mutagenic role for metabolically derived formaldehyde in E. coli. Mouse-liver S9 also enhanced the cell-killing effect of methylene chloride in the uvrA, and a recA/uvrA double mutant (AB2480) which is very sensitive to DNA damage. This pattern was consistent with formaldehyde damage. However, a mutagenic role in bacteria for the glutathione conjugate of methylene chloride cannot be ruled out by these E. coli experiments because S9 fractions did not increase 1,2-DBE mutagenicity, suggesting lack of cell wall penetration by this reactive species. Rat-liver S9 did not activate methylene chloride to a bacterial mutagen or enhance methylene chloride-induced cell-killing, which is consistent with the carcinogenicity difference between the species.

Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and/or functional role.

The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA glycosylase, abasic site nicking, and deoxyribose excising activities. Analysis of the amino acid sequence of this protein suggests that the Fpg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys motif. Competition experiments show that the Fpg protein substitutes Cu(II), Cd(II), and Hg(II), metal ions classically associated with substitutions in zinc finger proteins. The Fpg protein activities are inhibited following the reaction with a Cys-specific reagent at low protein:reagent ratios, suggesting that these residues are important for the enzymatic activities. Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys-->Gly mutations. Substitution of the zinc in these proteins by 65Zn(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger mutants. The mutations in the Fpg protein outside the zinc finger consensus sequence do not eliminate the Fapy-DNA glycosylase and abasic site nicking. One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites. Cys-->Gly mutations in the zinc finger consensus sequence reduce all three aforementioned activities substantially. The purified Fpg proteins with Cys-->Gly mutations in the zinc finger consensus sequence do not incise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein. The wild type Fpg protein and the Fpg proteins mutated outside the zinc finger sequence bind an oligonucleotide with a unique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg proteins within the zinc finger sequence do not bind to the same oligonucleotide. Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to DNA as well as decreased enzymatic activities.

Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver. Relationship to the Km and Vmax for nitrosamine metabolism.

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli.

Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups. In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites. Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases. The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination. The release of 5'-terminal dRp by crude extracts of E. coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.

Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase.

Self-complementary oligodeoxynucleotides have been synthesized containing O6-methylguanine (O6meG), O6-ethylguanine (O6etG), O6-isopropylguanine (O6iprG) and O4-methylthymine (O4meT). They anneal in solution to give double-stranded DNA. These double helices have been used as substrates for the DNA repair protein O6-alkylguanine-DNA-alkyltransferase coded for by the ada gene of Escherichia coli. The repair followed second-order chemical kinetics. O6meG was repaired by the 19-kd transferase at a rate of 2.54 x 10(7) M-1 s-1 which is close to the theoretical limit for a diffusion-controlled reaction; O6etG and O4meT are repaired 1,000 and 10,000 times more slowly. The 39-kd alkyltransferase (which is precursor to the 19-kd form) and the 19-kd transferase repaired O6etG at similar rates. O6iprG was not repaired. The repair of oligomers containing O6meG was only slightly inhibited by the presence of nonalkylated oligomers. Oligomers containing O6etG were only slightly more effective as inhibitors of repair than the nonalkylated oligomers, indicating that the transferase does not bind selectively to alkylated DNA. Parallel structural studies have shown that O6-alkylguanine:C and O4-alkylthymine:A base pairs have a similar geometry with the alkylated base displaced into the major groove of the DNA in contrast to O6-alkylguanine:T and O4-alkylthymine:G base pairs which retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine. Measurement of the rate of repair of these different base pairs suggests that pairs with the alkyl group exposed in the major groove may be repaired more rapidly than those with the alkyl group more deeply buried in the helix.

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC Working Paper No. 15/6. Effect of ethanol on nitrosamine metabolism and distribution. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence.

Alcohol consumption is associated with an increase in the incidence of cancers of several sites, including oesophagus, larynx and mouth. The mechanism of the induction of cancer by alcohol is not clear. Humans are exposed to a variety of carcinogenic N-nitroso compounds. Ethanol changes the pharmacokinetics of nitrosamines in rats particularly by decreasing the ability of the liver to metabolize them. A hypothesis is put forward that the influence of alcohol on human cancer is mediated by its effect on the metabolism and distribution of nitrosamines from the diet, from tobacco smoke and from endogenous synthesis.

Repair of synthetic oligonucleotides containing O6-methylguanine, O6-ethylguanine and O4-methylthymine, by O6-alkylguanine-DNA alkyltransferase.

32P-Labelled self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, and O4-methylthymine have been synthesized and used as substrates for the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AAT). The reaction was second-order with rate constants of 2.6 X 10(7) M-1 sec-1 for O6-methylguanine, 2.6 X 10(4) M-1 sec-1 for O6-ethylguanine and 2.5 X 10(3) M-1 sec-1 for O4-methylthymine. These oligomers should allow sensitive and specific assay of the mammalian enzyme.

Intra-abdominal pressure responses of mineworkers to standard loads.

Intra-abdominal pressure (IAP) has been used as the basis for a series of force limits for manual handling based on a limiting pressure criterion of 12 kPa (90 mm Hg). Measurements of the IAP responses to a series of standard loads were carried out on a group of eight instructors from the British coal mining industry. The results showed that there was a very low probability (.0016 or less) of obtaining an IAP value of 12 kPa or greater from the population represented by our sample. Average responses were 33% lower than would have been expected from previous reports on force limits based on IAP measurements. While more data are necessary in order to confirm the applicability of these findings to the general mining population, the results clearly suggest that greater loads could safely be lifted by mineworkers than can be lifted by the industrial population represented by the guidelines.