An effective algorithm for the serological diagnosis of idiopathic inflammatory myopathies: The key role of anti-Ro52 antibodies.

BACKGROUND: Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests. METHODS: Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods. RESULTS: Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA. CONCLUSIONS: Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended.

Multicentric evaluation of a second generation assay to detect antiviral citrullinated peptide antibodies: a collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni.

AIMS: A novel immunoenzymatic assay using viral citrullinated peptides derived from Epstein-Barr virus-encoded proteins (viral citrullinated peptide 2 (VCP2)) has been developed and evaluated by means of a multicentre collaborative study. METHODS: Three hundred nine sera from patients with established rheumatoid arthritis (RA), 36 with early arthritis, 12 with juvenile arthritis and 453 controls were tested for VCP2 and cyclic citrullinated peptide (CCP) antibodies. RESULTS: The VCP2 assay showed 78.3% sensitivity and 97.1% specificity. VCP2 and CCP had a high concordance rate in patients with RA (88%) and controls (97%). However, 36 RA sera were positive in the CCP assay but negative on VCP2, and two RA sera reacted only on VCP2. CONCLUSIONS: The new VCP2 assay is endowed with high sensitivity and specificity. VCP2-positive RA sera are mostly but not completely contained in the CCP-positive population. Studies are in progress to establish whether the VCP2 assay can detect clinically distinct subsets of patients with RA.

Evaluation of the effect of Bosentan treatment on proinflammatory cytokine serum levels in patients affected by Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular and fibrotic changes in the skin and in internal organs. Endothelin-1 (ET-1) is a peptide that has a role in promoting both vascular injury and the fibrotic process in SSc; indeed, patients with systemic sclerosis have higher levels of ET-1 compared with healthy subjects. Moreover, ET-1 enhances expression of pro-inflammatory cytokines in animal model. Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension and digital ulcers in scleroderma patients. In animal models and in vitro models, after treatment with Bosentan, a significant reduction of cytokine (TNF α, IFN γ,IL-8, IL-4) levels was observed. The aim of the study is to verify whether Bosentan treatment in SSc patients can reduce circulating cytokines levels. We enrolled 10 patients affected by SSc with digital ulcers and/or pulmonary hypertension, treated with Bosentan 125 mg twice daily. Patients were tested for cytokines and ET-1 level before treatment and after 12 months. The cytokines tested were IL-10, IL-2, IL-4, IL-5, IL-6, IL-8, GM-CSF, IFN-γ and TNF. Levels of ET-1, IL-10, IL-4, IL-5, GM-CSF and TNFalpha did not show consistent modification during treatment with Bosentan in respect to baseline, while IL-2, IL-6, IL-8 and IFN-γ were significantly decreased. Bosentan significantly reduced IL-2, IL-6, IL-8 and IFN- γ levels in SSc patients, probably slowing progression to fibrosis and vascular damage. This is the first report showing a decrease of profibrotic and proinflammatory cytokines levels in humans during treatment with Bosentan.

PARP activity and NAD concentration in PMC from patients affected by systemic sclerosis and lupus erythematosus.

The enzyme poly(ADP-ribose) polymerase (PARP-1, EC 2.4.2.30) is activated by DNA strand breaks caused by several agents and utilizes NAD to form polyADPR, bound to acceptor proteins. The involvement of PARP-1 in autoimmune diseases has been suggested: antiPARP autoantibodies are described in systemic lupus erythematosus (SLE), DNA strand breaks have been evidenced in systemic sclerosis (SSc). We tested poly(ADP-ribosyl)ation activity and NAD concentration in PMC from patients affected by SLE or SSc and from controls. Lower PARP-1 activity and higher NAD concentration were observed in pathological conditions than controls, supporting the role of PARP-1 activation in modulating NAD concentration.

[Viral genotype and HLA class II alleles influence on extra-hepatic manifestations of chronic HCV infection]. / Associazione tra genotipo virale, HLA e manifestazioni extra-epatiche dell'epatite C.

OBJECTIVE: To test whether an association between HCV genotype, HLA class II alleles distribution and extra-hepatic manifestations (EHM ) can be demonstrated in a group of Italian patients with chronic HCV infection . METHODS: Sixty patients affected by HCV infection with EHM were consecutively enrolled. 163 HCV patients without EHM were tested as controls for the prevalence of HCV genotypes, while we referred to literature as to the controls for HLA distribution. HCV-RNA was quantified by a RT-PCR. HLA class II alleles typing was performed using a standard microlymphocytotoxicity assay. We used chi-square or Fisher test (p<0.05 significant). Odds Ratio (OR) was performed by 2X2 contingency table. RESULTS: HCV 2c genotype was found in 63.46% of patients compared to 19.63% of controls (p<0.0001; OR=7.11). Furthermore, it correlated with carpal tunnel syndrome (p=0.03; OR=4.5) and autoimmune thyroiditis (p=0.02; OR=9.2). On the contrary, 1b genotype protected from EHM in toto (p=0.0004; OR=0.21) and particularly from carpal tunnel syndrome (p=0.0014; OR=0.07). Moreover, 3a genotype prevented HCV people from having cryoglobulinemia (p=0.05; OR=0.11). As to HLA, DR6 seemed to facilitate EHM in HCV patients (p=0.041; OR=1.61), while DQ2 (p=0.03; OR=0.5) and DQ3 (p=0.002; OR= 0.5) may play a protective role. In addition, HLA DR3 was associated with cryoglobulinemia (p=0.02; OR=9.5). CONCLUSIONS: According to our findings, 2c genotype can be considered as a major risk factor for developing HCVrelated EHM, while 1b genotype seems to prevent their onset; there are also evidences suggesting that HLA might play a role in chronic HCV infected patients.

Validation of a new immunoenzymatic method to detect antibodies to RNA polymerase III in systemic sclerosis.

OBJECTIVE: To test the reliability of a new enzyme-linked immunosorbent assay (ELISA) to identify anti-RNA polymerase III (RNAP III) positive sera from Italian patients with Systemic Sclerosis (SSc) and other chronic inflammatory disorders. METHODS: A comparison between the new ELISA for anti-RNAP III and the gold standard technique, immunoprecipitation (IP), was first performed on 106 SSc patients, 16 patients with other connective tissue diseases and 10 healthy subjects. A further ELISA evaluation was performed on 224 SSc patients, 120 subjects with other rheumatic or infectious diseases, and 81 healthy controls. RESULTS: Plotting ELISA and IP data in a Receiver Operator Characteristic curve, the ELISA cut-off value providing the best specificity (99.1%) and sensibility (100%) was 28 U/ml (AUC=0.999; p<0.0001). Using this cut-off in the second analysis, anti-RNAP III positive results were found in 41 (18.3%) SSc patients, all negative for anticentromere or anti-topoisomerase I antibodies, while only 3 subjects tested positive among the 120 sera collected from other patients. All the healthy subjects were negative. CONCLUSION: This new ELISA for anti-RNAP III is highly accurate when a proper cut-off value is employed and represents a valid substitute to IP in a clinical setting.

Antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. A collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni (FIRMA).

OBJECTIVE: Prothrombin (PT) is a target for antibodies with lupus anticoagulant (LA) activity, suggesting the possible application of anti-prothrombin antibody (aPT) assays in patients with antiphospholipid syndrome (APS). Different methods - both homemade and commercial - for the detection of aPT are available, but they seem to produce conflicting results. The purpose of this study was to compare the performance of different assays on a set of well-characterized serum samples. PATIENTS AND METHODS: Sera were gathered from 4 FIRMA institutions, and distributed to 15 participating centres. Forty-five samples were from patients positive for LA and/or anticardiolipin antibodies (aCL) with or without APS, and 15 were from rheumatoid arthritis (RA) patients negative for antiphospholipid antibodies. The samples were evaluated for IgG and IgM antibodies using a homemade direct aPT assay (method 1), a homemade phosphatidylserine-dependent aPT assay (aPS/PT, method 2), and two different commercial kits (methods 3 and 4). In addition, a commercial kit for the detection of IgG-A-M aPT (method 5) was used. RESULTS: Inter-laboratory results for the 5 methods were not always comparable when different methods were used. Good inter-assay concordance was found for IgG antibodies evaluated using methods 1, 3, and 4 (Cohen k > 0.4), while the IgM results were discordant between assays. In patients with thrombosis and pregnancy losses, method 5 performed better than the others. CONCLUSION: While aPT and aPS/PT assays could be of interest from a clinical perspective, their routine performance cannot yet be recommended because of problems connected with the reproducibility and interpretation of the results.

Low serum level of COMP, a cartilage turnover marker, predicts rapid and high ACR70 response to adalimumab therapy in rheumatoid arthritis.

The aim of this study was to evaluate serum biomarkers, used in clinical routine, to predict the American College of Rheumatology (ACR) response to long-term anti-TNF alpha treatment (adalimumab). Sera from 29 consecutive rheumatoid arthritis patients were analysed for anti-cyclic citrullinated peptide (anti-CCP), cartilage oligomeric matrix protein (COMP) and IgM and IgA RFs (class-specific rheumatoid factors) at the start of treatment with adalimumab and after 3, 6 and 12 months. The response to the therapy was evaluated by ACR 20, 50, 70 and by DAS 28 scores. The mean serum COMP level of the population did not change after treatment. However, patients with low serum COMP levels (<10 U/l) at baseline showed a significant (p<0.02) higher ACR70 response (>50%) within 3 months, and also at 6 months, than patients with higher COMP values (ACR70<20%). This was also reflected by significantly higher decrease in DAS score at 3 (p<0.02) and 6 months (p<0.01) treatments. The IgM RF titre decreased significantly (p=0.02) after the therapy, but the percentage of serum positivity for anti-CCP and IgA/IgM RF did not change. No significant correlation was shown between serum COMP levels and C-reactive protein/erythrocyte sedimentation rate during the follow-up. Neither were any correlations shown between ACR/DAS 28 scores and anti-CCP, Ig M/IgA RFs. Our data indicate that low (<10 U/l) serum COMP before starting anti-TNF alpha treatment predicts a rapid (within 3 months) and high ACR70 response compared to RA patients with higher COMP values. This might reflect different mechanisms in the cartilage process in the RA disease at that time of treatment with different therapeutic sensitivity to anti-TNF alpha treatment.

Association of extrahepatic manifestations with HLA class II alleles and with virus genotype in HCV infected patients.

It has been postulated that host factors, such as the human leucocyte antigen (HLA) system, may play a predominant role in the pathogenesis of HCV-related extra-hepatic manifestations. This study was performed to investigate the role of HLA- DR and DQ alleles in a group of Italian patients, with HCV infection and associated extrahepatic manifestations and to test whether an association between HCV genotype, HLA locus and clinical or serological manifestations can be demonstrated. Thirty unrelated patients affected by HCV infection with extra-hepatic manifestations were consecutively included in the study. One hundred and sixty-three HCV patients without extrahepatic manifestations were tested as controls for the prevalence of HCV genotypes, and 283 healthy donors were used as controls for HLA class II alleles distribution. HCV-RNA was quantified by an reverse transcription-PCR. HLA class II alleles typing was performed using a standard microlymphocytotoxicity assay on B lymphocyte purified. HCV 2c genotype was found in 53.3% compared to 18.4% of controls (p=0.00001; OR=5.1). Cryoglobulins were detected in 72.7% DR6+ patients and in 31.6% DR6- patients (p=0.05; OR=3.21). Rheumatoid factor was found in 90.9% of DR6+ patients and in 42.1% DR6- patients (p=0.018; OR 13.7). Only two DR5+ patients (20%) had cryoglobulinemia, while 6 patients (30%) in the DR5- group had cryoglobulinemia (p=0.02; OR=0.07). Associations were found between DR7 and ANA (OR=1.74) and between DQ2 and ANA (OR=1.97). According to our findings HLA-DR6 might play an important role in developing extra-hepatic manifestations and genotype 2c could be considered as a risk factor for their onset.

Relationship among parameters of lake polluted sediments evaluated by multivariate statistical analysis.

In the attempt to assess the relationship and interdependency among sediment toxic pollutants, in particular heavy metals, polycyclic aromatic hydrocarbons (PAH), and linear alkyl sulfonates (LAS) and some of the sediment typical components: inorganic carbon (IC), organic material (OM) and acid volatile sulphides (AVS), multivariate techniques of statistical analysis have been applied to a set of chemical data obtained by the analysis of the sediments of the Trasimeno Lake, a central Italy lake characterized by a large surface (128 km(2)) and a low mean depth (about 4.5 m). The results of principal component analysis (PCA) show interrelationships between: OM content and PAH, Pb, and Cu concentrations of the sediments, LAS and AVS, and AVS and IC. The effect of the different sampling periods on sediment composition and contamination level, and the clustering of the sampling sites as a consequence of pollutant load are also shown. The principal component bi-plot of the variables and samples indicates that PAH have the greatest influence on the separation of samples in the different sampling periods.

HLA-DPB1 alleles association of anticardiolipin and anti-beta2GPI antibodies in a large series of European patients with systemic lupus erythematosus.

Our objective was to determine the HLA-DPB1 allele associations of anticardiolipin (aCL) and anti-beta2GPI (a(beta)2GPI) antibodies, and of clinical manifestations of the antiphospholipid syndrome (APS), in systemic lupus erythematosus (SLE). We studied 577 European patients with SLE. aCL and a(beta)2GPI antibodies were measured by ELISA. Molecular typing of HLA-DPB1 locus was performed by polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. aCL showed positive association with -DPB1*1501 (P = 0.005, OR = 7.4), and -DPB1*2301 (P = 0.009, OR = 3.3). a(beta)2GPI showed positive association with -DPB1*0301 (P = 0.01, OR = 1.9), and -DPB1*1901 (P = 0.004, OR = 8.1). In addition, livedo reticularis was associated with -DPB1*1401, and Raynaud's phenomenon with -DPB1*2001. In conclusion, HLA-DPB1 locus may contribute to the genetic predisposition to develop antiphospholipid antibodies and clinical manifestations of the APS in patients with SLE.

Dosage and characterization of circulating DNA: present usage and possible applications in systemic autoimmune disorders.

The discovery of extracellular nucleic acids in the circulation was firstly reported in 1948. In the last few years it has been demonstrated that the entire spectrum of genetic changes seen in primary tumors could also be detected in the serum of patients with solid tumors. This observation has also opened up exciting possibilities for tumor detection and monitoring. More recently investigators started looking for other forms of non-host DNA in the plasma/serum so that in 1997 the presence of fetal DNA in the plasma/serum of pregnant women was demonstrated. This finding suggested that maternal plasma fetal DNA would be a very valuable material for noninvasive prenatal diagnosis and monitoring. It has been also postulated that the presence of the two-way trafficking of nucleated cells and free DNA between the mother and fetus may have potential implications for the development of certain autoimmune diseases. Concerning autoimmune disorders, Tan was the first author to describe the presence of high levels of circulating DNA in patients with systemic lupus erythematosus (SLE) in 1986. Later on different authors demonstrated that elevated levels of serum DNA was also present in patients with other diseases including rheumatoid arthritis. We have analyzed both circulating free DNA and DNA extracted from nucleated blood cells in scleroderma and in lupus patients but, by using gel electrophoresis, we were able to define the pattern of the DNA, instead of simply dosing its amount in the circulation. We have found that SLE and SSc have anomalous patterns of DNA both in serum and in the Buffy-coat and that these patterns are typical for each disorder. It is possible that understanding the biological significance of the diversity in DNA pattern exhibition in white blood cells may give new insights into the pathophysiology of autoimmune disorders. It is also conceivable that circulating and immune-competent cellular DNA markers might offer the promise of precise quantitative analysis useful for diagnostic purposes, without the need to establish difficult cutoffs as is necessary for protein markers.

[Chromosome aberrations, valued as frequency of spontaneous micronuclei, in subjects with suspected presclerodermic Raynaud's phenomenon]. / Anomalie cromosomiche, valutate come frequenza di micronuclei spontanei, in soggetti con fenomeno di Raynaud sospetto presclerodermico.

OBJECTIVE: To evaluate the prevalence of spontaneous chromosome damage in cultured peripheral lymphocytes of subjects with suspected presclerodermic Raynaud's phenomenon (RP), by means of molecular cytogenetic analysis. METHODS: We studied 20 suspected presclerodermic RP, 20 idiopathic RP and 25 healthy subjects. As marker of chromosome alteration we used the micronucleus assay. All subjects were also classified as ANA-, ACA+ or Scl70+. To identify the mechanism of MN formation, a MN fluorescence in situ hybridisation (FISH) analysis using a pancentromeric DNA probe was also performed. RESULTS: Suspected presclerodermic RP subjects, showed significantly higher MN frequencies than idiopathic RP and controls (39+/-15.2 vs 10+/-2.1 and 9.8+/-3.5 respectively p<0.0001). Interestingly, subjects with idiopathic RP displayed MN frequency comparable to that of controls. Furthermore, ACA+ subjects showed the highest MN frequencies (44+/-8.1) as compared to subjects with different antibody pattern (26+/-7.1). CONCLUSIONS: Our results show the presence of higher levels of chromosomal damage in circulating lymphocytes of suspected presclerodermic RP. They also would suggest a key role of anti-centromere antibody in determining the observed cytogenetic anomalies. FISH analysis indicated that both aneuploidogenic and clastogenic events contribute to the formation of MN observed in suspected presclerodermic RP.

Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis.

Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were approximately 50 and approximately 750 micromol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 micromol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50-100 micromol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.

Modification of DNA patterns in plasma and nucleated blood cells from systemic sclerosis patients.

OBJECTIVE: To analyze the DNA patterns extracted from plasma and nucleated blood cells (lymphocytes) in systemic sclerosis (SSc) with a new MFC DNA extracting kit. METHODS: Ten SSc patients and 9 healthy controls were studied. Heparin containing blood samples were separated into plasma and buffy coat fractions and subjected to DNA extraction. The DNA pattern was revealed by 0.4% agarose electrophoresis and analyzed in a Gelblot Programme file (UVP Product). RESULTS: In control samples the DNA pattern observed in plasma extract was different from that of the buffy coat. For the plasma a series of peaks ranging from 2-23 Kb were present, and for the buffy coat we usually observed 2 to 3 principle bands, respectively, at around 33 Kb and 0.5 Kb. For SSc patients the DNA patterns that resulted from the plasma and buffy coat were totally different from the control samples, with some exceptions. CONCLUSION: We observed that SSc samples contain a distinctively different DNA pattern compared to healthy controls. Further studies are needed to establish whether or not this DNA pattern might be considered peculiar to SSc, and whether or not the method is a useful tool for pathogenic studies of the disease and for diagnostic purposes.

Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells.

Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa.

Synthesis and structure--mutagenicity relationship of benzo-annulated cyclopentaphenanthrenes.

The synthesis of 2,3-dihydro-1H-indeno[5,4-a]anthracene (2), the fluoreno[a]anthracenes 3 and 4, 2,3-dihydro-1H-cyclopenta[a]chrysene (6), 3,4-dihydro-2-vinylphenanthrene (10) and cyclopenta[c]chrysenes 11, 12 has been described. Structure analysis of the new products by (1)H and (13)C NMR spectroscopy is presented. Estimates of the mutagenic activity of compounds 2--4, 6 and 11--14 in Salmonella typhimurium determined by Ames' test indicate that all products are inactive for both TA 98 and TA 100 strains except 4,5-dihydro-3H-cyclopenta[c]chrysene (12). The mutagenic properties of these compounds have been compared with those shown by previously studied benzo[g]cyclopenta[a]phenanthrenes and cyclopenta[c]phenanthrenes and discussed. Some conclusions have been drawn about the effects of benzoannulation and of the carbonyl function on the mutagenicity of this class of compounds.

Influence of culture conditions on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells.

The DNA-damaging ability of benzene and its metabolites on peripheral blood mononuclear cells (PBMC) has been investigated by using the alkaline comet assay. The PBMC were incubated with different compounds in two different media for 2 and 24 hr at concentrations that did not affect cell viability and the DNA damage was quantified by a computerized image analysis system. Benzene and phenol (5 mM) did not show any genotoxic activity after 2 hr of incubation in the two media tested, phosphate-buffered saline (PBS) and RPMI containing 5% of heat-inactivated fetal calf serum (RPMI + 5% FCS), whereas phenol was genotoxic and cytotoxic at 10 mM after 24 hr of incubation in RPMI + 5% FCS. All other benzene metabolites were genotoxic at micromolar concentrations when incubated in PBS with the following decreasing order of potency: benzenetriol, catechol, hydroquinone, and benzoquinone. When the PBMC were incubated in RPMI + 5% FCS, the effect of catechol (200-600 microM) and benzenetriol (10 microM) was reduced, whereas the genotoxicity of benzenetriol at high concentrations (50-100 microM) and hydroquinone (150-2500 microM) was not affected. In contrast, the effect of benzoquinone at 5 and 10 microM was greatly enhanced when the cells were incubated in RPMI + 5% FCS. This effect resulted mainly from the presence of serum in the medium and it was almost completely inhibited by boiling the serum (100 degrees C, 5 min) and was partially reduced by extensive dialysis. Benzoquinone was the most damaging compound when tested under more physiological conditions, thereby supporting the general observation that it is the most myelotoxic benzene metabolite.