Hans Joachim Seidel

Early effects of benzene exposure in mice. Hematological versus genotoxic effects

Female BDF1 mice were exposed to 100, 300 and 900 ppm benzene 6 h/day, 5 days/week, up to 8 weeks. Hematological studies included peripheral blood data, T4 and T8 lymphocyte counts in the blood and the spleen, hemopoietic stem and progenitor cell assays in the marrow (CFU-S, CFU-C, BFU-E, CFU-E). The single cell gel assay (“comet assay”) was applied in parallel with cells from the peripheral blood, bone marrow, spleen and liver. The results showed minor changes in the stem and progenitor cells and the development of a slight anemia at 4 and 8 weeks, in agreement with reported data. New was the increase of the T4/T8 ratio in the peripheral blood (not in the spleen) at the end of the first week of exposure to 300 and 900 ppm. The results of the “comet assay” indicate a much higher sensitivity of this test system (strand breaks and alkali labile sites of DNA). The tail moment indicative of the damage to DNA increased as early as 3 days with 300 ppm in the peripheral blood cells. Furthermore, the liver cells did react to a much higher extent than the other cells tested. With 100 ppm significant changes were seen in the liver after 5 days, but not in the blood. The repair, studied 24 and 48 h after the end of the exposure, was almost complete after 5-day exposure period in the blood and the liver, but not after 4 weeks of exposure with 300 ppm in the blood, and 100 and 300 ppm in the liver.

Determination of benzene metabolites in urine of mice by solid-phase extraction and high-performance liquid chromatography

A method was developed for quantitative measurement of trans,trans-muconic acid, catechol, hydroquinone and phenol in urine. Hydrolysis of esterified and glucuronized phenolic compounds was effected by specific enzymes. The hydrolysed mixture was purified and separated by solid-phase extraction with an anion exchanger, followed by extraction with diethyl ether. By using a clean-up procedure the natural background from mouse urine could be reduced, so that the detection limit of the metabolites was in the range 3-60 mg/l. Optimization of the chromatographic conditions resulted in a short high-performance liquid chromatography analysis time. Phenol had the longest retention time of about 10 min. The clean-up procedure could also be used for phenylmercapturic acid, an additional benzene metabolite, but for sensitive high-performance liquid chromatographic detection of phenylmercapturic acid other conditions are necessary.

Laser pyrolysis products - genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.

Action of benzene metabolites on murine hematopoietic colony-forming cells in vitro.

Five benzene metabolites--phenol, catechol, hydroquinone, parabenzoquinone, and trans,trans-muconaldehyde--were tested in vitro in murine hematopoietic cell cultures for erythrocytic (BFU-E and CFU-E) and granulocytic (CFU-C) colony growth. The dose range was 4 x 10(-4) to 4 x 10(-8) M. Of these compounds, phenol showed the lowest toxicity. In general, CFU-E cultures were far more sensitive than CFU-C and BFU-E. The results are discussed in relation to the test system used, the in vivo sensitivity of CFU-E, and the lack of knowledge about in vivo concentrations of these compounds.

Hematotoxic effects of benzene analyzed by mathematical modeling

The hematopoietic cell response to benzene intoxication in mice (during and after long-term inhalation) was analyzed by a mathematical model of murine hematopoiesis. Two complementary methods, Time-Curve and Steady-State Analysis, were developed to identify target cells for benzene toxicity and to quantify the extent of damage in different stages of development of these target cells. We found that (i) erythropoietic cells were the most sensitive; (ii) granulopoietic cells were about half as sensitive as erythropoietic and (iii) hematopoietic stem cells exhibited a sensitivity that ranged between that of erythropoietic and granulopoietic cells. A dose-response relationship between benzene levels and damage in target cells (valid from 1 to more than 900 ppm) was derived that was linear for doses up to 300 ppm and plateaued thereafter. This relationship indicated that benzene-induced hematotoxicity is subject to a saturable process. Recovery of hematopoiesis following chronic benzene intoxication was simulated for different doses and preceding exposure periods. The impaired recovery following exposure periods greater than 8 weeks could be explained by a severe reduction in the maximum self-maintenance of stem cells. This study indicates that the present mathematical model represents a useful approach to investigate alternate hypotheses for the action of hematotoxic agents.

Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

SummaryT cell leukemias were induced by a single dose of methylnitrosourea (MNU) in DBA/2, C57/Bl/6, NMRI, BDF1, and CBA mice. The latency period in the CBA strain was much longer than in the others. Studies on the pluripotent stem cells in the bone marrow and T cell reactions of thymus and spleen cells showed a toxicity of MNU for these parameters but no significant differences between the strains. The activity of the natural killer (NK) cells in the spleen and peritoneal exudate cells, studied also after additional stimulation by injection of Corynebacterium parvum, was influenced by MNU, but again no relation to leukemogenesis could be established. The first leukemic (transplantable) cells were found in the thymus. The presence of leukemic cells could be responsible for low NK cell activities found in BDF1 and DBA/2 mice late after MNU.

The influence of ethanol on the stem cell toxicity of benzene in mice.

BDF1 mice were exposed to 100, 300, and 900 ppm benzene vapor, and the numbers of hematopoietic progenitor cells, early and late erythroid progenitors (BFU-E and CFU-E) and granuloid progenitors (CFU-C), were determined with and without additional exposure to ethanol (5, 10, 15 vol%) in the drinking water. The duration of benzene inhalation was up to 4 weeks, 6 hr per day, 5 days per week. It was shown that the number of CFU-E per femur was depressed in a dose-dependent manner by benzene alone and also by ethanol combined with a given benzene concentration. CFU-E showed rapid regeneration after the end of the exposure, but not BFU-E and CFU-C. Prolongation of the ethanol exposure after withdrawal of benzene had only a marginal effect on progenitor cell regeneration.

Ferrokinetics and erythropoiesis in mice after long-term inhalation of benzene.

SummaryFerrokinetics and erythropoiesis were examined in mice exposed for 6 or 7 weeks to an airborne concentration of 300 ppm of benzene, for 6 h per day, and 5 days per week. Ferrokinetic indicators showed only a slightly enhanced production of haeme and erythrocytes in the spleen (133% ± 18% and 122% ± 17%, respectively). Production did not change in the femoral marrow; a decline of CFU-C, BFU-E and especially CFU-E (34% ± 8%) took place there and a shift of cellularity into less mature developmental classes in the erythroblast compartment, without this compartment as a whole being damaged. The erythrocytes produced have an enhanced MCV (109% ± 0%) and MCH (109% ± 1%) with an unchanged MCHC; their concentration in blood sank to 87% ± 1%. The absolute reticulocyte count rose to 160% ± 16%.59Fe incorporation into the liver declined far below the level attributable to decreased accessibility of the tracer (84% ± 4%). A shortening of the life span of late erythroblasts and circulating erythrocytes was deduced from these findings and methodological problems related to some of the seemingly controversial findings are discussed.

T-cell phenotypes in chemically induced leukemia in mice

T-cell leukemias were induced in adult BDF mice by a single i.v. injection of 50 mg kg-1 of methylnitrosourea (MNU). Leukemic cells from the thymus and other organ sites showed the theta antigen and were peanut-negative, but were heterogeneous with respect to Lyt-1 and Lyt-2. The acute cytotoxic effect of the MNU showed no preferential toxicity to a special T-cell subset in the thymus. During the latency period of leukemogenesis a continuous fall in peanut-positive cells in the thymus was found.

Effects of radiation on murine T-cell leukemogenesis induced by butylnitrosourea

SummaryBDF1 mice were exposed to butylnitrosourea for 12 weeks (BNU, 0.02% in the drinking water) and died of thymic lymphomas with median latency periods of 12–20 weeks. In addition to BNU, groups of mice received weekly radiation doses of 0.0625–1.0 Gy; 12×0.25 Gy enhanced leukemogenesis, 12×0.75 Gy delayed it and 12×0.50 Gy had no effect. Lower doses had marginal enhancing effects. After a dose of 12×1.0 Gy, the mice died earlier than after treatment with BNU alone and, as with 12×0.75 Gy, some extrathymic lymphomas were observed. The numbers of CFU-S in the femur and the spleen showed a dose-dependent depression, in addition to the effect of BNU alone. In lymphocyte stimulation assays with Con A und LPS and also in the mixed lymphocyte reaction, a reduced proliferation was found, again dependent on the radiation dose. Therefore, there was no correlation of leukemogenesis and the degree of stem-cell reduction or depression of these immune parameters. The delay of leukemogenesis by 12×0.75 Gy in addition to BNU may be caused by an enhanced target cell kill.