Search

Unlabelled: Radiation synovectomy is an effective treatment for chronic synovitis refractory to pharmacological treatment in patients with rheumatoid or seronegative arthritis. Concerns persist about possible radiation-induced cytogenetic damage after radiation synovectomy leading to recommendations to use this technique only in the elderly. Micronucleus (MN) frequency in lymphocytes and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8OHdG) as an indicator of cellular oxidative DNA base damage are biomarkers of radiation-induced cytogenetic damage. The course of both biomarkers was studied in patients with different types of chronic synovitis undergoing radiation synovectomy with very short-lived 165Dy-ferric-hydroxide (DFH)., Methods: Radiation synovectomy of the knee was performed in 13 men and 12 women (mean age, 44+/-15 y) using a mean activity of 9.48+/-1.65 GBq 165Dy-DFH in 27 consecutive treatments. MN frequency in lymphocytes and urinary excretion of 8OHdG, measured by high-performance liquid chromatography, were assessed before and 4 (MN only) and 20 h after radiation synovectomy., Results: Urinary excretion of 8OHdG in patients (in micromol/mol creatinine; pretreatment mean, 3.1+/-3.4; median, 2.27) was not significantly different from that in healthy volunteers (mean, 2.0+/-1.2; median, 1.87) and not altered by radiation synovectomy (post-treatment mean, 2.5+/-1.5; median, 2.04, NS). An increase in 8OHdG levels after radiation synovectomy of more than 1 SD was found in only 1 patient, who experienced leakage to the lymph nodes but who already had elevated urinary 8OHdG levels before treatment. The frequency of MN/500 binucleated cells (BNCs) was slightly lower in patients (pretreatment mean, 4.3+/-2.6; median, 4.25) than in healthy volunteers (mean, 5.4+/-2.3; median, 5.3) and did not significantly change after therapy, either (4-h post-treatment mean, 3.9+/-2.1, median, 3.8; 20-h post-treatment mean, 4.1+/-2, median 3.8 MN/500 BNC). In 22 of 27 treatments, no leakage to nontarget organs could be monitored, whereas leakage to the local lymph nodes and the liver was detected after 5 treatments., Conclusion: Radiation synovectomy using 165Dy-DFH causes no significant radiation burden to most patients as indicated by the absence of adverse changes in levels of biomarkers of cytogenetic damage and a low incidence of leakage. These data suggest that the risk of malignancy may not be elevated.

The purpose of this study was to examine the molecular mechanism responsible for the defective insulin-stimulated glucose transport in cultured fibroblasts from a patient (VH) with clinical features of Werner syndrome and severe insulin resistance. Thus, in cells derived from VH, the subcellular distribution, structure, functional activity, as well as plasma membrane insertion of GLUT1 glucose transporters were analyzed. Furthermore, the insulin signal transduction pathway leading to activation of phosphatidylinositol (PI) 3-kinase as well as components of GLUT1-containing membrane vesicles were characterized. In fibroblasts derived from VH, GLUT1 glucose transporters were overexpressed by 8-fold in plasma membranes (PM) and by 5-fold in high density microsomes, respectively. Exofacial photolabeling revealed that only 14% of the overexpressed PM-GLUT1 transporters were properly inserted into the plasma membrane. The complementary DNA structure of the patient's insulin receptor and the GLUT1 glucose transporter, the intrinsic activity of plasma membrane glucose transporters, the tyrosine phosphorylation, as well as the protein expression of insulin receptor substrate-1/2 and p85 alpha/beta- and p110 alpha/beta-subunits of PI 3-kinase were normal. However, insulin-stimulated association of the p85 subunit of PI 3-kinase was defective in fibroblasts derived from VH compared to those from controls, and this defect was associated with a reduced IRS-1-dependent activation of PI 3-kinase by 50.2% and 63.6% after incubation for 5 and 10 min with 100 nmol/L insulin, respectively. Furthermore, immunodetection of small GTP-binding Rab proteins in subcellular membrane fractions indicated a decreased expression of Rab4 in total cellular homogenates as well as in high density microsomes by 70% and 58%, respectively. After preparation of GLUT1-containing vesicles, Rab4 was not detected to be a component of these vesicles. Analysis of the PI 3-kinase in GLUT1-containing membrane vesicles revealed insulin-dependent targeting of the p85 subunit to the vesicles immunoadsorbed from VH and control fibroblasts. Importantly, the association of the p85 subunit as well as the p85-immunoprecipitable PI 3-kinase activity were markedly reduced in GLUT1-vesicles derived from the patient. In conclusion, impaired PI 3-kinase activity in GLUT1-containing membrane vesicles derived from fibroblasts of VH is associated with a defective docking and/or fusion process of glucose transporters with the plasma membrane and thus might contribute to the molecular defect causing insulin resistance in this patient.

Genotoxicity related to waste anaesthetic gas exposure is controversial. We have investigated the frequency of sister chromatid exchanges in peripheral lymphocytes of operating room personnel exposed to trace concentrations of isoflurane and nitrous oxide. Occupational exposure was recorded using a direct reading instrument. Frequencies of sister chromatid exchanges were measured in lymphocyte cultures of 27 non-smokers working in the operating room and 27 non-smoking controls. Personnel were exposed to an 8-h time-weighted average of nitrous oxide 11.8 ppm and isoflurane 0.5 ppm. After exposure, sister chromatid exchange frequency was increased significantly (mean 9.0 (SD 1.3) vs 8.0 (1.4) in exposed and control personnel, respectively) (P < 0.05). We conclude that exposure to even trace concentrations of waste anaesthetic gases may cause genetic damage comparable with smoking 11-20 cigarettes per day.

The question of whether or not inhalation anaesthetics are genotoxic remains controversial. Therefore, we have studied the in vitro genotoxic potential of isoflurane and nitrous oxide in human lymphocytes. Blood samples were obtained from eight healthy male, non-smoking volunteers, which were incubated and exposed to increasing concentrations of isoflurane (0.0, 0.3, 0.6 and 1.2 mmol litre-1) or 50% nitrous oxide in oxygen. Baseline sister chromatid exchange (SCE) rate per cell was mean 7.65 (SD 1.5) which increased to 9.15 (1.0), 9.55 (1.4) and 9.95 (1.8) SCE/cell during exposure to isoflurane 0.3, 0.6 and 1.2 mmol litre-1, respectively. During 50% nitrous oxide exposure, SCE rate was 9.26 (1.4). The difference between the control and exposed cells was statistically significant (P < or = 0.05). We conclude that exposure to nitrous oxide and subanaesthetic concentrations of isoflurane can produce genetic damage in peripheral lymphocytes in vitro.

The phenomenon of spontaneous increased micronuclei and enhanced UV-sensitivity, which is known for familial cutaneous malignant melanoma (CMM) patients, could be demonstrated again in fibroblasts of 17 familial CMM patients. In order to determine if close relatives of familial CMM patients show both a comparable spontaneous chromosomal instability and enhanced UV-sensitivity, cultured fibroblasts of 24 healthy, first-degree relatives of patients with familial malignant melanoma were investigated. The cytokinesis-block micronucleous technique was used to detect enhanced chromosomal instability. Fibroblasts of the investigated relatives showed a significantly increased spontaneous formation of micronuclei, in comparison to 19 healthy controls, but no enhanced UV-sensitivity was evident. We conclude that chromosomal instability might be a hereditary trait and a causative factor in developing familial malignant melanoma. This supports the concept of a genetic predisposition to familial CMM and may help to identify high-risk family members at a cytogenetic level in addition to the common clinicopathological traits.

The action of insulin and IGF-1 in comparison to non-diabetic controls was studied in cultured fibroblasts of a patient with an inherited syndrome of insulin resistance (Type A syndrome). Insulin binding was reduced due to decreased receptor affinity, but sequence analyses revealed no alterations of splicing or primary insulin receptor (IR) structure. Most likely due to the IR affinity defect analyses of signal transduction pathways showed an impairment of insulin action on glucose uptake, total RNA synthesis and phosphorylation as well as activity of MAP-kinase. In addition inducibility of c-fos mRNA level was strongly impaired by insulin and IGF-1, but comparable to controls by PDGF indicating a postreceptor defect. In conclusion, we provide evidence that genetic syndromes of insulin resistance can be associated with both, receptor and postreceptor defects.

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2'-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2'-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2'-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.

The energy demand during physical exercise causes an increased oxygen uptake and supply to active tissues, which may increase the rate of free oxygen radical production and thereby affect the capacity of endogenous cellular defense systems. This could result in DNA base modifications, among which 8-hydroxydeoxyguanosine (8OHdG) is one of the most important and has widely been used as a biomarker of in vivo oxidative lesions. Therefore, we examined the effect of regular running exercise on the urinary levels of 8OHdG in 32 long-distance runners and in a group of untrained healthy subjects. The range of 8OHdG in urine was 0.12-6.45 mumol/mol creatinine in both groups, and no significant difference in the mean excretion levels between runners and control probands was observed. This gives no reason to believe that physical exercise in trained individuals may induce a disturbance of the oxidant-to-antioxidant balance.

In a preliminary study Bamezai and Kumar (1992) reported that a 24-h period of sleep deprivation may raise sister chromatid exchange (SCE) frequencies up to 193% in peripheral blood lymphocytes. This was reinvestigated to clarify the role of sleep duration as a confounder for SCE, which is a well-established parameter for biomonitoring in occupational medicine. In our study, the SCE baseline and the influence of a 24-h period of sleep deprivation (test period) on SCE were investigated for 20 non-smoking volunteers (10 females and 10 males; 20-29 years of age). There was no significant difference (Pall = 0.094) between the deviations of the two SCE rates of the control period (mean: -0.21 +/- 0.90 SCE) and the differences between SCE rates before and after sleep deprivation (mean: 0.42 +/- 0.94 SCE) of each proband. No significant difference was detected between females and males, and SCE did not correlate with age or sleep duration. Therefore we conclude that the influence of sleep deficit on SCE is in the range of a normal day-to-day variance, and has not to be taken into account when SCE is used for a genotoxic monitoring at the workplaces.

To determine if low-level exposure to lead has an effect on arterial blood pressure, demographic and clinical data of a group of 507 males without any occupational exposure to lead were analyzed in a retrospective study. The following variables were included in the analysis: age, height, weight, nicotine consumption, alcohol intake, blood lead, systolic blood pressure, diastolic blood pressure, and erythrocyte count. The effects of the variables age, weight, height, alcohol intake, nicotine consumption, and blood lead on blood pressure were investigated by stepwise linear regression analysis. The diastolic as well as the systolic blood pressure was significantly influenced by Body Mass Index, age, and alcohol. A significant effect of lead could be found only for diastolic blood pressure. None of the possible interactions were significant enough to merit inclusion in the statistical model. Regarding the strength of the effects on diastolic blood pressure we find that lead ranks after age, weight. In conclusion, this study shows that lead has an effect on diastolic arterial blood pressure in a survey of a group of middle-aged, occupationally nonexposed men.

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn462-a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser462 mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, Thus, like the previously described Glu460 mutation, the Ser462 mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 microM MNNG. The detection limit for the assay was 5 microM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmokers controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (CO-Hb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the structure and function of the insulin receptor in a patient (PK) with severe insulin resistance and Rabson-Mendenhall syndrome. Insulin binding to cultured fibroblasts from PK was almost not detectable and insulin-induced insulin receptor autophosphorylation and glucose uptake was abolished. The structure of the receptor gene was analysed by sequencing amplified products of the 22 exons with the flanking intron regions directly as well as after subcloning in pUCBM20 plasmids. Two mutant alleles of the insulin receptor gene were detected. One allele contains in-frame 12 additional base pairs in exon 3 coding for the amino acids Leu-His-Leu-Val located between Asp-261 and Leu-262 in the receptor's extracellular domain, being the first report of an insertion mutation of the insulin receptor gene. In the other allele Arg-86 in exon 2 is changed into a stop codon. Therefore, PK is compound heterozygous at the insulin receptor locus. Direct cDNA sequencing indicates that both mutant alleles are expressed in the patient's fibroblasts. Studies of the parents' fibroblasts revealed that PK inherited the insertion mutation from the father and the nonsense mutation from the mother. Insulin binding to fibroblasts of the mother was reduced (63% of control cells) and hormone binding to the father's cells shows a larger reduction (37% of control cells), but less severe than the patient's cells (11% of control). This investigation provides further evidence that the Rabson-Mendenhall syndrome is causally related to mutations in the insulin receptor gene.

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Two groups of workers occupationally exposed to glycol ethers in a varnish production plant or the ceramic industry were examined. For 19 persons the external and internal exposure was assessed on the Monday and Tuesday after an exposure-free weekend. In the varnish production area the concentrations of 2-ethoxyethanol (EE), 2-ethoxyethyl acetate (EEAc), and 2-butoxyethanol (BE) in air averaged 2.9, 0.5, and 0.5 ppm, respectively, on the Monday, and 2.1, 0.1, and 0.6 ppm, respectively, on the Tuesday. At the same workplaces the mean urinary 2-ethoxyacetic acid (EAA) and 2-butoxyacetic acid (BAA) concentrations were 53.2 and 0.2 mg/l on Monday preshift and 53.8 and 16.4 mg/l on Tuesday postshift. The results show that glycol ethers are very well absorbed through the skin. Therefore biological monitoring is indispensable. To study the kinetics of the toxic metabolite, 17 persons were examined for their excretion of EAA in urine during an exposure-free weekend. The median values of the calculated half-times were 57.4 and 63.4 h, respectively, which are longer than the values presented in literature until now. According to our calculations the limit value should not exceed 50 mg EAA per liter of urine, which is the current German biological tolerance value (BAT value) for EAA in urine. The maximum concentration value at the workplace (MAK value) for EE and EEAc in air should be revised. Finally, the subjects from the varnish production plant as well as a group of reference persons were studied for cytogenetic effects of glycol ethers (sister chromatid exchange, micronucleus test). Such effects could not be detected.

A modified assay for the detection of DNA single-strand breaks (SSBs) in human mononucleated white blood cells (MWBCs) based on the nick translation (NT) reaction was developed and combined with the test for unscheduled DNA synthesis (UDS). Both assays were performed on disposable 96-well filtration plates and therefore allowed rapid and sensitive examination of SSBs and UDS. Only 5-8 ml of heparinized blood is required for an eightfold determination in both assays. The uptake of radioactive nucleotide precursors was demonstrated to depend linearly upon the NT reaction time and in both assay systems on the number of investigated cells. The best results and the lowest signal to noise ratio were obtained when the NT assay was performed at 25 degrees C for 20 min. The test was standardized for 150,000 MWBCs/well and a polymerase I concentration of 20 U/ml. The same number of cells were used to measure UDS during a 4-h incubation at 37 degrees C. We observed a dose-dependent increase in SSBs after in vitro incubation with N-methyl-N-nitrosoguanidine (MNNG), with a detection limit of 50 microM when MNNG was present for 1 h and of 5 microM after 20-h incubation period. UDS in MWBCs was increased after treatment for 1 h with MNNG (200 microM) only if poly(ADP)ribose synthesis was inhibited by 3-aminobenzamide. UDS was induced by 320 microM methyl methanesulfonate, but SSBs could only be detected after inhibition of UDS by 100 microM hydroxyurea.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated a family in which at least 4 men in 3 generations had a syndrome of obesity, mild mental retardation, delayed puberty, macroorchidism, acanthosis nigricans, hyperinsulinemia, and later overt insulin-resistant diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM). The patients have markedly curly scalp hair, deficient face and body hair. Their teeth were healthy and normal in size and position. The clinical and biochemical findings and characteristics of the insulin receptors investigated in fibroblasts are reported. There was normal insulin binding to fibroblasts in the 2 brothers and their father. However, insulin-stimulated RNA synthesis was decreased as compared to that of normal control individuals. These findings suggest a postbinding defect of insulin action. The pedigree documents an autosomal dominant mode of inheritance. The diagnosis is of practical importance since it enables medical supervision of gene carriers in a preclinical state of atherosclerotic complications and overt diabetes. The findings in this family have relevance also to the explanation of familial mild mental retardation and to the study of different forms of insulin resistance due to a disturbance in biosignal transfer.

Fibroblast cultures of 16 basal cell epithelioma (basalioma, BCE) patients with an unusually young age at onset of disease (29-51 years; 42.5 +/- 7.04), and healthy normal controls (27-55 years; 40.73 +/- 9.52) were studied for chromosome instability induced by ultraviolet rays (UV). We used an UV source that emitted predominantly UV-A and UV-B at an intensity of 375 J/m2 and evaluated the induction of micronuclei (MN) and sister chromatid exchange (SCE). Young basalioma patients and normal controls showed no significant differences in MN and SCE frequencies, neither with respect to spontaneous nor to UV-induced values (MN spontaneous: 10.80 +/- 5.65 vs. 11.32 +/- 8.21; UV-induced increase: 7.36 +/- 4.40 vs. 9.93 +/- 7.55; SCE spontaneous: 10.28 +/- 1.61 vs. 10.72 +/- 1.09; UV-induced increase: 7.30 +/- 2.19 vs. 7.55 +/- 2.14). We conclude from these data that an enhanced UV sensitivity as observed in cells from patients with cutaneous malignant melanoma and xeroderma pigmentosum is not a constitutive risk factor in basalioma patients.

The induction of micronuclei was studied in human diploid fibroblasts incubated in the presence of the tobacco-specific nitrosamine NNK. We used four fibroblast strains having a high capacity of O6-alkylguanine DNA alkyltransferase (13.0-23.3 pmol O6-methylguanine repaired per 8 x 10(6) cells) and four strains that showed no detectable repair capacity. Incubation with NNK doubled the frequency of micronuclei in repair-deficient cells but failed to evoke any effect in the proficient cell strains. Control experiments were performed with the direct methylating agent MNNG and in the presence of inhibitors of either metabolic activation or alkyltransferase. The results showed that the genotoxicity of NNK is dependent on the relationship between its metabolic activation and the constitutive DNA repair. This supports earlier findings that low constitutive levels of O6-alkylguanine DNA alkyltransferase may increase susceptibility to lung cancer after exposure to DNA methylating agents.

The induction of micronuclei (MN) by incubation with different mutagenic agents was tested in diploid fibroblast cultures obtained from 15 probands with constitutively high MN rates (15.75-77.25 MN/500 cells; average 36.27 +/- 17.60 MN) and 15 probands (controls) with low MN rates (4-13.75 MN/500 cells; average 8.97 +/- 2.73 MN). In order to find out whether fibroblast cultures of individuals with increased spontaneous MN levels exhibit an increased sensitivity to various agents with different genotoxic mechanisms, we studied the induction of MN in these cell cultures by ultraviolet (UV) irradiation, mitomycin C (MMC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo-(a)pyrene-diol-expoxid (BPDE). In addition, we tested aphidicolin (APC), a polymerase alpha inhibitor, which is a potent inducer of common fragile sites. Probands with spontaneously high MN showed an significantly increased sensitivity to UV (p less than or equal to 0.005), MMC (p less than or equal to 0.005), and BPDE (p less than or equal to 0.005). No significant differences were found for MNNG and APC as compared to controls.

Cultured fibroblasts from patients with cutaneous malignant melanoma (CMM) were tested for chromosomal instability by determination of micronuclei (MN) and sister chromatide exchange (SCE). The constitutive as well as the UV-induced level of MN was increased in CMM patients, being most pronounced in the familial cases. There was an increased UV sensitivity in CMM by using SCE, but no spontaneously enhanced SCE value. The enhanced UV sensitivity in CMM patients was in a comparable range as those of XP heterozygotes. We thus conclude that chromosomal instability is a concurrent feature of a genetic predisposition for CMM.

The in vitro formation of benzo[a]pyrene-DNA adducts was determined in peripheral blood monocytes of 22 lung cancer patients with at least one first-degree relative with lung cancer and compared to results obtained in 30 healthy controls. In patients, the mean (SEM) adduct formation was 2.8 (0.3) fmol/micrograms DNA as compared to 2.1 (0.1) fmol/micrograms in controls (p less than 0.05), and it was independent of age and smoking habits. These findings support the hypothesis that carcinogen-DNA adduct formation may be one factor of a constitutionally enhanced lung cancer risk.

During 3 years of collaboration we have detected 13 patients with insulin resistance syndromes among the 800 patients referred to our genetic counseling center for diagnosis during that time. It represents about 1.5% of all diagnostic problems of our genetic counseling center. Thus it seems to us that insulin receptor disorders are not as rare as previously appreciated. We describe here the clinical and biochemical findings of four of them as well as characteristics of disorders of insulin receptors. On cellular level these four patients represents the full scale of insulin receptor defects and demonstrate genetic heterogeneity of these disorders.

It was the aim of this study to compare the formation of DNA adducts in different human white blood cells. Lymphocytes and monocytes were isolated from peripheral blood by density centrifugation and by a monocyte-specific antibody linked to magnetic beads. DNA adducts were determined by 32P-postlabelling. After in vitro incubation with benzo[a]-pyrene (B[a]P) we found DNA adducts in monocytes, but not in unstimulated blood lymphocytes. Only after growth stimulation by phytohaemagglutinin did lymphocytes show an adduct pattern similar to monocytes. In contrast, B[a]P-7,8-dihydro-9,10-diolepoxide, an activated intermediate of B[a]P metabolism leads to similar DNA adducts in both cell types. When lymphocytes were incubated in the presence of B[a]P together with monocytes, then after subsequent separation lymphocytes exhibited most of the adducts found in monocytes, though to a lower extent. Our results suggest that unstimulated blood lymphocytes--which are unable to activate metabolically promutagens like B[a]P--receive the genotoxic material to form DNA adducts from other blood cells or from cells in the vessel lining. We conclude that the amount and the pattern of DNA adducts formed in whole white blood cells may be influenced considerably by a variable proportion of monocytes.

The frequency of kinetochore (centromere)-positive micronuclei (MN) was determined in 32 fibroblast cell lines. We tested 16 probands with spontaneously high MN levels (greater than or equal to 20 MN/500 cells (4%] and 8 probands (controls) with low MN levels (less than or equal to 13 MN/500 cells (2.6%]. To study whether the elevation of MN levels is due to increased chromosomal breakage we used the antikinetochore antibody fluorescent staining method. Probands with spontaneously high MN had kinetochore-positive MN increased by a factor 2.1 compared to the controls whereas the kinetochore-negative MN were increased by a factor 6.14. This shows that spontaneous elevation of MN is mainly caused by increased chromosomal breakage and only in a minor proportion by chromosome segregation errors as a consequence of spindle defects.

Cultured fibroblasts of 17 first-degree relatives of familial melanoma patients and six first-degree relatives of cutaneous melanoma (CMM) patients with multiple CMM primaries were tested for in vitro sensitivity to UV light. Fibroblasts of nine familial CMM patients with a known UV-sensitivity and 19 healthy probands served as a control. Sister chromatid exchange (SCE) was used as a parameter to detect UV-induced genotoxic damage. We found significantly (p less than 0.001) increased UV-induced SCE levels in familial melanoma patients, as well as in first-degree relatives of familial melanoma patients (p less than 0.001) after UV-A,B irradiation (375 J/m2), compared to the healthy probands without a family history of CMM. A significant (p less than 0.001) increase of UV-induced SCE was also observed in the relatives of CMM patients with multiple CMM primaries. In addition, the spontaneous SCE were significantly increased (p less than 0.05) in familial CMM patients. This study shows that increased UV sensitivity is a familial phenomenon. It is consistent with the concept of a genetic predisposition to CMM, which is based on increased UV sensitivity and may help to define groups with an elevated risk of developing cutaneous malignant melanoma.

Not a single agent is known to cause an increase of genetic disorders in humans. Even studies on large numbers of children born to parents after exposure to ionizing radiation or DNA alkylating agents failed to detect significant genetic consequences. This is in contrast to effects observed in human somatic cells or germ cells, and to various investigations with laboratory animals. A definite explanation of these discrepancies does not exist. Nevertheless, on the basis of the currently available human data it cannot be justified to advise a previously exposed person against having children, if this individual is otherwise healthy. There are obvious incongruencies between the potency of an agent to cause mutations at all, and to induce genetic effects in the offspring. It should therefore be emphasized that a regulatory classification of agents according to a potential genetic hazard in man must not solely be based on in vitro mutagenicity data.

Spontaneous micronuclei (MN) were determined in 69 fibroblast lines in the first subculture after cryoconservation. 45 cultures (65%) showed micronuclei in the normal range (less than 10 MN/500 cells), but 24 (35%) exhibited an elevation up to 60 MN/500 cells. In order to determine whether freezing and thawing is responsible for the enhanced MN level we studied the persistence of elevated MN in 10 cell cultures after freezing and thawing, and found that the MN levels returned to normal after 3 subcultures. In addition, the micronucleus formation before and after freezing was investigated in 2 newly established cell cultures obtained from probands whose cells had a particularly high number of MN. Both cultures had regular MN levels before freezing but a more than doubled number of MN when frozen samples were recultivated. This effect of the freezing procedure was confirmed with 5 separate biopsies obtained from the same donor. Since a freezing-related increase in MN was constantly observed in cells from some individuals but not in others, it might be possible that it is a constitutive trait which causes cultured fibroblasts of some individuals to be hypersensitive to the freezing and thawing procedure. It is also noteworthy that fibroblasts from 8 out of 12 probands with a familial malignant melanoma exhibited this phenomenon.

The frequency and distribution of rare and common chromosomal fragile sites in metaphases derived from peripheral lymphocytes were compared in 26 patients with malignant solid tumours and 24 normal controls. In order to avoid bias in evaluation, the identity of each individual as patient or control was disclosed only after the study was completed. Rare heritable folic acid inducible fragile sites were found in five patients (2q13; 6p23; 8q22; 16p12) and two controls (8q22). Common fragile sites were present in 21 of 26 patients and in 19 of 24 controls. These differences are statistically not significant in the Fisher test. We conclude that the expression of fragile sites does not indicate a predisposition for solid tumours.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.

In a controlled study, ten male volunteers were subjected to different smoking and passive smoking conditions. After 60 h of strictly controlled nonsmoking, five smokers were exposed to mainstream smoke only, while five nonsmokers were exposed to the gas phase of environmental tobacco smoke (ETS). In a second experiment smokers were mainstream and ETS exposed, while nonsmokers were exposed to complete ETS. Blood was drawn before and after smoking and DNA adducts were analysed from blood monocytes by the 32P-postlabelling assay, using the nuclease P1 enhancement method. We detected DNA adducts in monocytes of all probands. These adducts unrelated to smoking showed interindividual differences but only minor intraindividual changes in four samples of the same donor. After smoking interindividually variable additional adducts were visible in active smokers only. These smoking-related adducts had disappeared after 40 h of nonsmoking and reappeared again in three out of five smokers after the second smoking period. We conclude that smoking causes an interindividually variable pattern of DNA adducts in active smokers. These adducts disappear in less than 2 d, owing to the fast turnover of monocytes in the intravascular system. The effects described could not be observed in heavily exposed passive smokers.

In inbred mice strain DBA/2, genetically controlled differences in aryl hydrocarbon hydroxylase (AHH) activity have been demonstrated that predispose to aplastic anemia. To test the hypothesis of a similar mechanism in humans, we studied the formation of benzo(a)pyrene DNA adducts and water-soluble metabolites in skin fibroblasts from eight patients with aplastic anemia and eight normal controls. The ratio of water-soluble metabolites and DNA adducts was 46.5 +/- 16.6 in patients and was significantly lower as compared to 82.9 +/- 38.5 in controls (P less than 0.05). We conclude that increased formation of genotoxic intermediates may be a pathogenetic mechanism in some patients with aplastic anemia.

Patients with Bloom's syndrome, an autosomal recessively inherited disorder, have a highly increased risk of developing early malignancies of various types. The chromosomes of such patients exhibit an enhanced rate of spontaneous sister chromatid exchange. Cocultivation of patients' fibroblasts (as responder cells) with fibroblasts from healthy donors or from patients with xeroderma pigmentosum or Fanconi's anaemia (as effector cells) produces a dose-dependent reduction in Bloom-specific sister chromatid exchange; however, Bloom heterozygotes exhibit a reduced corrective capacity. The reduction in Bloom-specific sister chromatid exchange is related to the presence of a soluble factor (m.w. 10 000-20 000) which is produced in culture by normal proliferating fibroblasts. This corrective factor might represent a cancer-protective principle that is defective in patients with Bloom's syndrome.

Formation of DNA adducts and of water-soluble metabolites was studied in monocytes of 86 first-degree relatives of 15 families. Tests were performed with blood monocytes using (G-3H)-benzo[a]pyrene as a model pro-carcinogen. Variance analysis revealed significantly higher inter-familial than intra-familial variations. From these data we conclude that the formation of DNA adducts is genetically controlled. Therefore the enhanced formation of benzo[a]pyrene DNA adducts in lung cancer patients found in earlier studies may reflect a genetic predisposition for lung cancer in some patients.

The effect on the rate of sister chromatid exchanges (SCEs) in Bloom syndrome fibroblasts by cocultivation with Fanconi anemia and xeroderma pigmentosum fibroblasts and with Bloom syndrome heterozygotes was studied. Cells of Fanconi anemia and xeroderma origin reduced the rate of SCEs in Bloom cells by about 45%-50%, just as control cells do. In contrast, heterozygous Bloom cells reduced the rate of SCEs by only 16%-28%. In absolute figures, Fanconi cells reduced the mean rate of SCE in Bloom cells from 55.7 +/- 5.50- to 27.7 +/- 6.44, xeroderma cells to 30.5 +/- 5.73, and control cells to 28.3 +/- 5.35. Three different cell strains from Bloom syndrome heterozygotes reduced the rate to 40.1 +/- 8.81, 47.0 +/= 6.94, and 47.5 +/- 8.32. There was no effect on any of these cell strains by Bloom syndrome fibroblasts. We interpret the functional deficiency of heterozygous Bloom syndrome fibroblasts as a gene dosis effect. It probably represents a specific manifestation of the yet unknown primary defect, because it suggests the existence of a "corrective factor" that is inactive or absent in homozygous Bloom cells and reduced in heterozygotes. It may be identical with or closely related to the normal gene product at the Bloom locus.

Microscopic slides were mounted with paraffin on Teflon plates that contain 18 holes of 6 mm diameter each. This arrangement is reversible and yields 18 individual cell culture wells on one slide, thus allowing a simultaneous culture of different cell lines under identical conditions. The glass-paraffin junction can be easily detached and the paraffin sticks exclusively to the Teflon.

Six strains of Bloom syndrome (BlS) fibroblasts responded to co-cultivation with normal fibroblasts at a 1:2 ratio by a reduced rate of sister chromatid exchanges (SCE's) from a mean of 67.5 (range = 59--78) to 28.4 (range = 21--35). The response was dose-dependent in one strain tested at 1:2, 1:1, and 2:1 ratios. In addition, quadriradial exchange figures and other signs of increased chromosomal instability were not found in BlS cells following co-cultivation with control cells. Control cells did not respond to BlS cells and maintained a normal rate of SCEs. Culture medium conditioned for 48 hrs by normal fibroblasts could also reduce the rate of SCEs in BlS fibroblasts, but less than in co-cultivation. We suggest that the reduced rate of SCEs and the lack of chromosomal instability in BlS cells following co-cultivation represent a corrective effect that is related to the basic defect and not dependent on cell-to-cell contact.

Cultured fibroblasts of homozygotes and heterozygotes for Bloom syndrome exhibit an enhanced formation of micronuclei. The number of spontaneously occurring micronuclei permit clear separation of heterozygotes from either normal controls or homozygous patients without overlap between these groups. The observed differences could not be enhanced further by the addition of various mutagens. We conclude from the increased chromosomal damage that heterozygotes for Bloom syndrome may have a higher risk for malignant diseases.

Benzo[a]pyrene 4,5-oxide was covalently bound to DNA of cultured human fibroblasts and caused sister chromatid exchange. The monooxygenase inhibitor alpha-napthoflavone suppressed this induction of sister chromatic exchange, but did not affect binding to DNA. Control experiments with 4-nitroquinoline 1-oxide showed that alpha-naphthoflavone does not inhibit sister chromatid exchange in general. A more likely explanation for the discrepancy between induction of sister chromatid exchange and binding to DNA is that benzo[a]pyrene 4,5-oxide itself can bind to DNA, but this binding does not lead to a significant increase in sister chromatid exchange. However benzo[a]pyrene 4,5-oxide can be oxidized by monooxygenase to yet unknown products which are potent inducers of sister chromatid exchange. An important conclusion from this is that a biological effect such as the induction of sister chromatid exchange may correlate with the exact nature of DNA binding rather than with total binding, to the point where just measuring total binding may be completely misleading if intended to detect the causes of the biological effect.

Cocultivation of fibroblast cells from a male patient with Bloom syndrome (BS) and a female control reduced the rate of sister chromatid exchanges in the BS cells from a mean of 54 SCE per metaphase (range 42--65) to 41 (range 24--59). Medium used to culture control cells for 48 h also reduced the rate of SCE (from 40--65 to 33-54), whereas medium used for only 24 h altered the SCE rate only slightly (to 39--61). Dialyzed medium concentrate with molecular cutoff at 15,000 did not alter the SCE rate. These initial studies suggest that normal cells produce an agent, presumably lacking in BS cells, that is capable of mitigating the chromosomal manifestation of the BS mutation (bl) in bl/bl cells.

Human diploid fibroblasts cultured in Dulbecco's Modified Eagle's medium (DME) were exposed to different concentrations of 15 antibiotics to determine the limiting toxic concentration. The number of cells surviving after antibiotic treatment was given as the index of toxicity. No visible chromosomal damage could be detected when half the maximal toxic concentration was applied. The maximum limiting concentration was found to be the same for both the preconfluent and postconfluent phases.