Retinal findings in systemic sclerosis: a comparison with nailfold capillaroscopic patterns.

OBJECTIVE: To determine the prevalence of retinal disease in systemic sclerosis (SSc) and to characterise the findings of retinopathy. Additionally, to analyse the association of retinal disease with other clinical/laboratory findings, particularly the findings of nailfold capillaries in patients with SSc. METHODS: Photographs of the ocular fundi were taken and were evaluated by an ophthalmologist who was unaware of the SSc status of the patients. The nailfold capillaries were analysed with a dermatoscope. Patients were divided into two groups according to the presence (group A) or absence (group B) of retinal disease. RESULTS: Retinal findings of the patients with SSc consisted of hard exudates, vascular tortuosity, microhaemorrhage, and macular degeneration. The prevalence of retinal disease among the patients with SSc was 34% (10/29), compared with 8%(3/38) among the controls (p=0.011). The mean systolic blood pressure and the age of the patients in group A were significantly higher than those in group B. However, there was no significant difference in the nailfold capillary damage between groups A and B. CONCLUSION: Retinal abnormalities are often seen in patients with SSc and they may reflect the vascular changes characteristic of SSc. However, retinal changes may differ in quality from the changes of nailfold capillaries.

Retinal disease in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the incidence of retinopathy in systemic lupus erythematosus (SLE) and to clarify its significance in relation to other clinical manifestations. METHODS: A cross sectional study on lupus retinopathy was made in 69 patients with SLE. One expert ophthalmologist examined the ocular fundi of the lupus patients without any information of their disease state. Clinical and laboratory findings in the patients with retinopathy and those without were compared. RESULTS: Retinopathy was found in 7/69 (10%) patients. The findings included haemorrhages, vasculitis, cotton wool spots, and hard exudates, all of which were considered to reflect vascular damage. Retinopathy was found to be associated with the presence of anticardiolipin antibody (p<0.05) and with central nervous system lupus (p<0.01). The patients with retinopathy had higher levels of serum creatinine than the patients without retinopathy (p<0.01). The disease activity of lupus, as assessed by the maximum SLE disease activity index (SLEDAI) score of the patients, was also significantly higher in the patients with retinopathy (p<0.03). CONCLUSION: Incidence of retinopathy in SLE was similar to that in previous reports and it may reflect tissue microangiopathy, particularly associated with vasculitis or anticardiolipin antibodies, or both.

The inhibition of phenylhydroquinone-induced oxidative DNA cleavage by constituents of Moutan Cortex and Paeoniae Radix.

Moutan Cortex (root cortex of Paeonia suffruticosa ANDREWS) and Paeoniae Radix (root of Paeonia lactiflora PALLAS) are crude drugs used in many traditional prescriptions and have constituents in common. We studied the effects of extracts of these crude drugs and their constituents on oxidative DNA damage caused by phenylhydroquinone (PHQ), a major metabolite of o-phenylphenol. Both drugs suppressed the cleavage of pUC18 DNA induced by PHQ, and scavenged the superoxide and hydroxy radical generated by the chemical. They also inhibited the oxidative DNA cleavage by tert-butylhydroquinone (TBHQ), one of the major metabolites of butylated hydroxyanisole. When constituents were examined with the same system, galloylpaeoniflorin and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) were found to be the most potent inhibitors of the DNA cleavage. These constituents had oxygen radical scavenging activity. Paeonol also attenuated the DNA cleavage. Paeoniflorin and albiflorin had relatively small inhibitory effects on DNA cleavage. However, catechin enhanced the PHQ-induced DNA cleavage. The suppression of oxidative DNA damage by Moutan Cortex and Paeoniae Radix might be attributable to the additive effects of galloylpaeoniflorin, PGG and other constituents.

Suppressive effects of Hochu-ekki-to, a traditional Chinese medicine, on IgE production and histamine release in mice immunized with ovalbumin.

We examined the effects of Bu-Zhong-Yi-Qi-Tang (Japanese name: Hochu-ekki-to, HET), a traditional Chinese medicine, on IgE production and histamine release in mice immunized intraperitoneally with a mixture of ovalbumin (OA) and aluminum hydroxide (alum adjuvant). Three groups of mice were orally administered 0, 1.7 or 17 mg of HET on day 13 after the first immunization with a mixture of 1 microg OA and 1 mg alum adjuvant. They were again immunized with the same dose of OA plus alum adjuvant on day 14. The immunological changes in mice treated with OA alone or OA plus HET were examined, and the following findings were obtained. In the HET-treated mice, the elevation of anti-OA IgE in serum, and histamine release from basophils in blood, were significantly suppressed. A significant suppression of interleukin-4 (IL-4) secretion and proliferation of splenic lymphocytes in primary culture was also observed. A tendency to suppress the elevation of anti-OA IgG1 in serum and interleukin-2 (IL-2) secretion from splenic lymphocytes was observed in the HET-treated mice. These findings suggest that oral administration of HET suppresses IgE antibody production and histamine release in type I allergic reaction in mice immunized with OA plus alum adjuvant; this shows the efficacy of HET in treating type I allergic diseases, such as asthma.

DNA cleavage and 8-hydroxydeoxyguanosine formation caused by tamoxifen derivatives in vitro.

DNA damage caused by tamoxifen and its derivatives was examined by estimating the conversion of supercoiled pUC18 plasmid DNA to linear form by means of agarose gel electrophoresis. N-Desmethyltamoxifen induced DNA cleavage and its effect was enhanced by the addition of reducing agents such as dithiothreitol, NADPH and 2-mercaptoethanol. 4-Hydroxytamoxifen itself had little effect, but the cleavage was slightly enhanced by the addition of reducing agents. DNA damage was higher with alpha-hydroxytoremifene than with alpha-hydroxytamoxifen, which had a prominent effect only at high concentration. The cleavage by alpha-hydroxy derivatives were not enhanced by reducing agents. No damage was induced by tamoxifen, toremifene, 3-hydroxytamoxifen or N-desmethyltoremifene. The DNA cleavage by N-desmethyltamoxifen was inhibited by the addition of EDTA, mannitol, sodium azide, methionine, catalase and superoxide dismutase. The formation of 8-hydroxy-2'-deoxyguanosine was also examined with calf thymus DNA in vitro. A slight increase of its level was found with 4-hydroxytamoxifen in the presence of dithiothreitol and also with N-desmethyltamoxifen in the presence of NADPH, but alpha-hydroxytoremifene and alpha-hydroxytamoxifen were ineffective. These experimental data suggest that among metabolites of tamoxifen, N-desmethyltamoxifen and probably also 4-hydroxytamoxifen cause oxidative DNA damage in which redox cycling is involved. The DNA damage by alpha-hydroxytoremifene appears to involve a different mechanism from that by N-desmethyltamoxifen. Tamoxifen and toremifene are possibly metabolized to the forms contributing to DNA damage.

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Inhibition of Na+,K(+)-ATPase by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, a major constituent of both moutan cortex and Paeoniae radix.

The inhibition of Na+,K(+)-ATPase activity by various constituents of Moutan Cortex and Paeoniae Radix was studied. 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose (PGG), a major component of both crude drugs, strongly inhibited Na+,K(+)-ATPase activity (IC50 = 2.5 x 10(-6) M), whereas galloylpaeoniflorin, benzoic acid, and catechin were weakly inhibitory, and albiflorin, oxypaeoniflorin, paeoniflorin, paconol, and phenol were ineffective. The inhibition of Na+,K(+)-ATPase activity by PGG was decreased in the presence of BSA or phospholipids. The inhibition mode of PGG was noncompetitive with respect to ATP. The K0.5 value for Na+ was increased by the addition of PGG from 9.1 to 12.3 mM, whereas that for K+ was not altered. PGG also inhibited K(+)-dependent p-nitrophenyl phosphatase activity with an IC50 value of 5.3 x 10(-6) M, and the extent of the inhibition increased at higher concentrations of K+. The K0.5 value for K+ was decreased by the addition of PGG from 3.3 to 2.0 mM. These results suggested that the inhibition of Na+,K(+)-ATPase activity is caused by interaction of PGG with the enzyme in the E2 state. The inhibitory effect of Moutan Cortex or Paeoniae Radix is considered to be mainly attributable to PGG.

Contribution of oxygen radicals to DNA cleavage by quinone compounds derived from phenolic antioxidants, tert-butylhydroquinone and 2,5-di-tert-butylhydroquinone.

The effects of synthetic phenolic antioxidants, tert-butylhydroquinone (TBHQ), 2,5-di-tert-butylhydroquinone (DTBHQ) and 3-tert-butyl-4-hydroxyanisole (BHA), on DNA cleavage were examined with supercoiled plasmid DNA, pUC18, in vitro. Extensive single and double strand breaks of DNA by TBHQ were observed and almost all the DNA was converted to the linear form at 10(-2) M. The cleavage was stimulated by both CuCl2 and FeCl2, though the effect of FeCl2 was smaller. Metal ion chelators and some oxygen radical scavengers inhibited the cleavage. The generation of TBHQ semiquinone radical and hydroxyl radical in the presence of copper was demonstrated by ESR spectroscopy. DTBHQ also caused DNA cleavage, though its effect was much smaller than that of TBHQ. BHA had no effect in the experimental systems employed. Oxygen radicals were considered to contribute to the DNA cleavage by TBHQ and DTBHQ.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated with tert-butylhydroquinone, a major metabolite of butylated hydroxyanisole.

Oxidative DNA damage caused by butylated hydroxyanisole (BHA), 2-tert-butyl(1,4)hydroquinone (TBHQ, a metabolite of BHA) and 2,5-di-tert-butyl(1,4)hydroquinone (DTBHQ), as well as 2,6-di-tert-butyl(1,4)benzoquinone (BHTQ, a metabolite of butylated hydroxytoluene), was evaluated by measuring the formation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymus DNA. 8OHdG formation was greatly increased by TBHQ in a concentration-dependent manner. This effect was strongly enhanced by CuCl2 and suppressed by EDTA, bathocuproinedisulfonic acid disodium salt, methionine, glutathione reduced form or catalase, but was not affected by mannitol, sodium benzoate or sodium azide. Thus, TBHQ-induced 8OHdG formation may be mediated by copper. DTBHQ also induced the formation of 8OHdG, though to a much lesser extent than TBHQ, and its effect was stimulated by CuCl2. BHA had a small enhancing effect at high concentration, only in the presence of CuCl2, whereas in the case of BHTQ, it occurred both in the presence of CuCl2 and FeCl2.

Specific inhibition of Na+,K(+)-ATPase activity by atractylon, a major component of byaku-jutsu, by interaction with enzyme in the E2 state.

Atractylon, a major component of the crude drug "Byaku-jutsu" (rhizomes of Atractylodes japonica), strongly inhibited Na+,K(+)-ATPase activity with an I50 value of 8.9 x 10(-6) M. It also inhibited Mg(2+)-ATPase, H+,K(+)-ATPase, H(+)-ATPase and Ca(2+)-ATPase activities, but less potently. No effects on alkaline and acid phosphatase activities were observed. The inhibition of Na+,K(+)-ATPase activity by atractylon was noncompetitive with respect to ATP and was greater with increasing K+ concentration, whereas it was not affected by Na+ concentration. The activity of K(+)-dependent p-nitrophenyl phosphatase, a partial reaction of Na+,K(+)-ATPase, was inhibited noncompetitively with respect to substrate (I50 value of 1.8 x 10(-5) M), and the inhibition rate was independent of the K+ concentration. Furthermore, atractylon increased the Ki value for Na+ from 130 to 190 mM, but did not alter the Ki value for ATP. Inhibition of the phosphoenzyme formation by atractylon was greater at 0.1 M than at 1 M NaCl. K(+)-dependent dephosphorylation (E2-P to K.E2) was inhibited by atractylon, whereas ADP-sensitive (Na.E1-P to Na.E1) and non-specific dephosphorylation steps were not affected. These results suggest that atractylon, a specific inhibitor of Na+,K(+)-ATPase, interacts with enzyme in the E2 state and inhibits the reaction step from E2-P to K.E2.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major metabolite of O-phenylphenol.

The generation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymus DNA treated with O-phenylphenol (OPP) or its major metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), was studied. The content of 8OHdG residues was increased in DNA treated with PHQ, and the generation of 8OHdG was highly dependent on PHQ concentration. PBQ had little effect on the formation of 8OHdG, and OPP had no effect. The formation of 8OHdG by PHQ was reduced by oxygen radical scavengers such as catalase, sodium benzoate and sodium azide. The PHQ-induced 8OHdG formation was accelerated by the addition of CuCl or CuCl2 to the reaction mixture, but was decreased by the addition of chelating agents such as EDTA, bathocuproinedisulfonic acid disodium salt (bathocuproine disulfonate) and O-phenanthroline. These results demonstrate that hydroxyl radicals generated in the process of oxidation of PHQ contribute to the formation of 8OHdG in DNA, and copper ions facilitate the oxidative DNA damage. Copper ions greatly accelerated the PHQ-induced DNA cleavage in vitro, although they had no effect on cleavage without PHQ. On the other hand, DNA cleavage occurred by the addition of FeCl2 in the absence and presence of PHQ. FeCl2 stimulates 8OHdG formation only slightly with or without PHQ. Furthermore, the stimulatory effect of FeCl2 on 8OHdG formation was observed even in the presence of EDTA. The formation of 8OHdG in bladder DNA is likely to be one of a series of events leading to bladder tumors seen in rats fed OPP-containing diet.

DNA cleavage by metabolites of butylated hydroxytoluene.

The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di-t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 x 10(-6) M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including superoxide dismutase (SOD), catalase, polyethylene glycol, t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl2. The production of superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro.

[U-14C]o-Phenylphenol (OPP) was found to bind covalently to calf thymus DNA during a 60 min incubation in the presence of microsomes, but not in their absence, indicating that metabolic conversion of the parent compound, OPP, to an activated form is essential. Postlabeling analysis with bladder DNA of rats fed a diet containing 2% OPP for 13 weeks revealed one major adduct on TLC. In an in vitro postlabeling experiment with calf thymus DNA, both of the major metabolites of OPP, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), formed adducts, but no adducts were observed with OPP. The chemical structure responsible for adduct formation is thought to be the PHQ semiquinone radical intermediate formed during interconversion between PHQ and PBQ. When the oligonucleotides, pd(A)12-18, pd(C)12-18, pd(G)12-18 and pd(T)12-18, were used in vitro, only pd(G)12-18 gave TLC-detectable adducts on treatment with PHQ and PBQ. The covalent binding appears to be rather specific to guanine residues. These results suggest that covalent binding of the OPP metabolite is one of the underlying events in OPP-induced carcinogenesis in rats.

Inhibition of Na+,K(+)-ATPase activity by beta-eudesmol, a major component of atractylodis lanceae rhizoma, due to the interaction with enzyme in the Na.E1 state.

beta-Eudesmol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), inhibited Na+, K(+)-ATPase activity most strongly among the various kinds of phosphatases examined. It also inhibited Ca(2+)-ATPase and H+, K(+)-ATPase, but to a lesser extent. Its effect on Mg(2+)-ATPase was minute. No effects on H(+)-ATPase or alkaline and acid phosphatase activities were observed. The effects of beta-eudesmol on horse kidney Na+, K(+)-ATPase were studied in detail, and the following results were obtained: (1) beta-eudesmol inhibited the Na+, K(+)-ATPase activity with an I50 value of 1.6 x 10(-4) M. The mode of its inhibition was uncompetitive with respect to ATP; (2) it prevented the stimulation of enzyme activity by Na+. The inhibition gradually increased in accord with the increase of Na+ concentration, and it was constant when Na+ was higher than 6.3 mM; (3) it did not alter the K+ concentration necessary for half-maximal activation (K0.5 for K+); and (4) it inhibited the enzyme activity with a mode of action different from ouabain. Phosphorylation of enzyme with [gamma-32P]ATP was inhibited by beta-eudesmol with an I50 of 1.4 x 10(-4) M. The inhibition was greater in 1 M NaCl than in 0.1 M NaCl. It had no effects on dephosphorylation steps, i.e. none of the non-specific, the ADP-sensitive (Na.E1-P----Na.E1) and the K(+)-dependent (E2-P----K.E2) dephosphorylation processes were affected. These results suggest that beta-eudesmol, a relatively specific inhibitor of Na+, K(+)-ATPase, interacts with the enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P.

Acute effects of intravenous nicardipine on hemodynamics and cardiac function in patients with a healed myocardial infarction and no evidence of congestive heart failure.

Acute effects of intravenous nicardipine (10 micrograms/kg) on systemic hemodynamics and cardiac function were evaluated in 17 patients with a healed myocardial infarction and no evidence of congestive heart failure. Mean New York Heart Association functional class was 1.6 +/- 0.5 (mean +/- standard deviation). Aortic systolic pressure (p less than 0.001) and left ventricular end-diastolic pressure decreased (10 +/- 3 to 8 +/- 3 mm Hg, p less than 0.01), and systemic vascular resistance decreased significantly (p less than 0.001), whereas pulmonary and right atrial pressure and pulmonary arteriolar resistance did not change. Cardiac and stroke indexes showed biphasic changes. Although positive and negative maximal rate of left ventricular pressures decreased significantly (p less than 0.05 and p less than 0.01, respectively), they did not change significantly when aortic systolic pressure was corrected. There was a significant inverse correlation between the negative rate of left ventricular pressure/aortic systolic pressure before nicardipine infusion and its maximal percent increase after infusion (r = -0.56, p less than 0.05), indicating a beneficial effect on diastolic relaxation in patients with impaired diastolic function. Our data show that a low dose (10 micrograms/kg) of intravenous nicardipine exerts a favorable effect on impaired diastolic function, but depresses left ventricular pump function with much less effect on right heart circulation.

Cardiorespiratory responses to standing arm ergometry in patients with ischemic heart disease. Comparison with the results of treadmill exercise.

To compare cardiorespiratory responses to standing arm ergometry and treadmill exercise, two graded exercise stress tests were performed in 30 patients with ischemic heart disease (IHD). Cardiac catheterization and expired gas analyses were also done. Standing arm ergometry was discontinued because of arm fatigue in 15 (50%) patients, whereas treadmill exercise was stopped due to leg fatigue in 8 (27%) patients. Maximal increase in rate-pressure product and oxygen uptake, and magnitude of ST-segment depression during standing arm ergometry were significantly smaller (p less than 0.01, p less than 0.01 and p less than 0.05, respectively) than those during treadmill exercise. Furthermore correlations of maximal change in rate-pressure product, oxygen uptake and extent of ST-segment depression were not close between the two exercise tests (r = 0.76, r = 0.67 and r = 0.54, respectively). Our results indicate that the ability to detect IHD with standing arm ergometry is lower than that with treadmill exercise and that it is not possible to predict accurately one's capacity for arm exercise from the treadmill exercise test.

[The effects of crude drugs using diuretic on horse kidney (Na+ + K+)-adenosine triphosphatase].

In the folk-medicine, several kinds of crude drugs are used as diuretics. Twenty three kinds of diuretic drugs were chosen, and examined for their effects on the horse kidney (Na+ + K+)-adenosine triphosphatase (ATPase), which is an intrinsic enzyme of the plasma membrane and responsible for the active transport of Na+ and K+ across the membrane. Twenty one out of twenty three kinds of ethanol extracts of diuretic drugs inhibited the kidney (Na+ + K+)-ATPase activity. The intensity of the inhibition of these drugs was compared by estimating the amounts of their ethanol extracts which inhibited the (Na+ + K+)-ATPase activity by 50% (I50, micrograms/ml). Among these drugs, Atractylodis Lanceae Rhizoma (I50 = 12.8) Atractylodis Rhizoma (I50 = 15.2), Plantaginis Semen (I50 = 16.0), Plantaginis Herba (I50 = 16.0) and Alismatis Rhizoma (I50 = 22.0), have strong inhibitory effects on the kidney (Na+ + K+)-ATPase activity. The ethanol extracts of the rhizomes of Atractylodes lancea De Candolle and Atractylodis japonica Kitamura were examined with varying concentrations of ATP and ouabain. The mode of inhibition of these two extracts on the (Na+ + K+)-ATPase activity appeared to be uncompetitive with respect to ATP as judged from Lineweaver-Burk plot. The ethanol extract of Atractylodes japonica Kitamura decreased the I50 for ouabain from 1.6 x 10(-7) to 7.0 x 10(-9) M, while that of Atractylodes lancea De Candolle did not change the I50 for ouabain.

Antigenic determinant of a monoclonal antibody: extracellular domain at the M3-M4 junction of the alpha-subunit of Na,K-ATPase.

The binding site of a monoclonal antibody, M45-80, against the alpha-subunit of horse Na,K-ATPase was determined. Various sizes of DNA fragments derived from rat Na,K-ATPase alpha 1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18. Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens. An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse alpha 1-subunits) at the M3-M4 junction located on the extracellular side. The ouabain-binding site is discussed in relation to the recognition site of M45-80.

DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol.

The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.