Bleomycin sulfate-induced micronuclei in human, rat, and mouse peripheral blood lymphocytes.

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 micrograms/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37 degrees C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species' concentration-response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations > or = 20 micrograms/ml. The nucleation index was significantly decreased in all species at either 80 or 160 micrograms/ml.

Inhalation studies of the genotoxicity of trichloroethylene to rodents.

Trichloroethylene (TCE) (CAS No. 79-01-6) is an industrial solvent used in degreasing, dry cleaning, and numerous other medical and industrial processes. Controlled inhalation studies were performed using male C57BL/6 mice and CD rats to determine if TCE can induce cytogenetic damage in vivo. Animals were exposed in groups of five to target concentrations of either 0, 5, 500, or 5000 ppm TCE for 6 h. Tissue samples were taken between 18 and 19 h post exposure. Peripheral blood lymphocytes (PBLs) in rats and splenocytes in mice were cultured and analyzed for the induction of sister-chromatid exchanges, chromosome aberrations, and micronuclei (MN) in cytochalasin B-blocked binucleated cells. Bone marrow polychromatic erythrocytes (PCEs) were analyzed for MN. The only positive response observed was for MN in rat bone marrow PCEs. TCE caused a statistically significant increase in MN at all concentrations, inducing an approximate fourfold increase over control levels at 5000 ppm. TCE was also cytotoxic in rats, causing a significant concentration-related decrease in the ratio of PCEs/normochromatic erythrocytes. This study indicates that there may be species-specific cytogenetic effects attributed to TCE inhalation exposure. In follow-up studies, CD rats were exposed for 6 h/day over 4 consecutive days to either 0, 5, 50 or 500 ppm TCE. No statistically significant concentration-related increases in cytogenetic damage were observed. While the MN frequencies in the 4-day study were comparable to those at the equivalent concentrations in the 1-day study, they were not significantly elevated due to an unusually high MN frequency in the controls. A subsequent replication of the 1-day 5000 ppm TCE exposure with rats again showed a highly significant increase in MN frequencies compared to concurrent controls.

Cytogenetic effects of phosphine inhalation by rodents. I: Acute 6-hour exposure of mice.

Phosphine (PH3) is a highly toxic grain fumigant that can be produced from the reaction of metal phosphides with water. To determine the in vivo cytogenetic effects of inhalation of PH3, male CD-1 mice were exposed to either 0, 5, 10, or 15 ppm target concentrations of PH3 for 6 hr. Twenty hours after the termination of exposure, the spleens of the mice were removed, macerated, and the splenocytes cultured for analyses of sister chromatid exchanges, chromosome aberrations, and micronuclei in cytochalasin B-induced binucleated cells. In addition, bone marrow smears were made for the analysis of micronuclei in polychromatic erythrocytes. No increase in any of the cytogenetic endpoints was found at any of the concentrations examined. The only statistically significant response was a concentration-related slowing of the cell cycle in the splenocytes.

DNA adducts and induction of sister chromatid exchanges in the rat following benzo[b]fluoranthene administration.

Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.

Cytogenetic studies of mice exposed to styrene by inhalation.

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.

Interspecies cytogenetic comparisons: studies with X-radiation and bleomycin sulfate.

A series of in vitro experiments were conducted to determine if there are innate differences in the sensitivity of peripheral blood lymphocytes (PBLs) from different mammalian species to clastogens. Mouse, rat, and human whole blood samples were exposed to either 0, 0.38, 0.75, 1.5, or 3.0 Gy x-radiation or 0, 5, 10, 20, 40, or 80 micrograms/ml bleomycin for 4 hr. Bromodeoxyuridine-containing cultures were initiated and the PBLs stimulated to divide with phytohemagglutinin. All cultures were harvested following a 3-hr colcemid treatment. Slides were made and differentially stained, and first-division metaphases were scored for chromosome aberrations. In the x-radiation studies human PBLs were significantly more sensitive than mouse PBLs which were in turn more sensitive than rat PBLs as measured by either the total percent aberrant cells or the number of dicentrics. Data from all three species could be fitted to a linear-quadratic model. Results with bleomycin suggest that the mouse and human PBLs are equally sensitive to the clastogenic effects of bleomycin. Both appeared to be more sensitive than the rat PBLs, but the variation between experiments was such that the results among species were not significantly different. These results indicate that there may be inherent differences in sensitivity among PBLs of mammalian species; however, more studies are needed to determine if the differences presented here hold for other agents.

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes.

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.

Cytogenetic studies of ethyl acrylate using C57BL/6 mice.

The clastogenicity of ethyl acrylate (EA) was examined in vivo by injecting i.p. five male C57BL/6 mice per dose group with either 125, 250, 500 or 1000 mg/kg EA dissolved in saline. Controls received solvent only. Acrylamide (100 mg/kg), because of its similarity in structure and mode of action to EA, was used as a positive control. Twenty-four hours after injection, the animals were anesthetized and the spleens aseptically removed. Splenocytes were isolated on density gradients and cultured with concanavalin A to stimulate cell division. In half the cultures bromodeoxyuridine was added at 21 h for analysis of chromosome aberrations (CAs) in first division cells and sister chromatid exchange (SCE) in second division cells. In the remaining cultures cytochalasin B was added to produce binucleated cells for scoring of micronuclei (MN). There was no significant increase in SCE or CAs at any of the doses of EA examined. At the highest dose examined (1000 mg/kg), EA did cause a small but significant increase in binucleated cell MN. Acrylamide caused an increase in MN and SCEs in splenocytes. Because others have found EA to be clastogenic in vitro, isolated splenocytes were exposed to a wide range of concentrations of EA during the G0 stage of the cell cycle or 23 h after mitogen stimulation during the late G1 or early S phase of the cell cycle. Although EA was toxic for both exposure regimens, significant increases in chromatid-type aberrations were found only when the target cells were treated 23 h after mitogenic stimulation. No statistically significant increase in SCE frequency was found after either treatment regimen. These data suggest that EA is only clastogenic at near toxic concentrations during a specific stage of the cell cycle.

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard.

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.

A comparison of sister-chromatid exchange in mouse peripheral blood lymphocytes exposed in vitro and in vivo to phosphoramide mustard and 4-hydroxycyclophosphamide.

Cyclophosphamide (CP) and two of its known metabolites, 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), were analyzed for their ability to induce sister-chromatid exchanges (SCEs) in mouse peripheral blood lymphocytes (PBLs) in vitro and in vivo. At equimolar concentrations, CP is a more potent SCE inducer in vivo than PAM and PAM and 4-OHCP induce equal numbers of SCEs in a dose-dependent manner. The present study also shows that these metabolites of CP are more potent SCE inducers than CP itself in vitro. This relationship might be explained by the differences in pharmacokinetics of these compounds.

Sister chromatid exchange induction by diaziquone in human and mouse lymphocytes following both in vivo and in vitro exposures.

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative currently being tested as an experimental chemotherapeutic agent, was used to treat mouse and human peripheral blood lymphocytes (PBLs) to determine its genotoxic potential by examination of sister chromatid exchange (SCE) induction. In vitro exposure to AZQ caused a linear increase in SCE in both mouse and human PBLs, with mouse PBLs being about twice as sensitive as the human cells. The lowest in vitro concentration found to induce a significant effect on SCE frequency was 0.3 micrograms/ml in mice and 1.0 micrograms/ml in human PBLs. Mice exposed by either i.p. or i.v. injection showed similar dose-related linear increases in SCE frequencies in their PBLs. After i.v. administration of AZQ, splenocytes from treated mice showed approximately the same SCE frequency as found in the PBLs. In general, AZQ caused a slowing of cell cycling in vivo while giving inconsistent responses in vitro. AZQ did cause a dose-related decrease in the number of recoverable mononuclear lymphocytes in mice treated in vivo. Contrary to the in vitro studies, comparison of SCE responses in mice with those previously observed in brain tumor patients undergoing chemotherapy with AZQ (Kligerman et al., Cancer Res., 47: 631-635, 1987) revealed AZQ was a much more potent SCE inducer in humans than in mice.

Persistence of SCE-inducing lesions in lymphocytes of mice exposed to diaziquone.

Male C57B1/6 mice were injected i.p. with either 1.25 or 5.0 mg/kg diaziquone (AZQ) and killed at various time intervals from 1 to 99 days post treatment for examination of sister chromatid exchange (SCE) persistence in the peripheral blood lymphocytes (PBLs) and splenocytes. SCE frequencies were found to decay steeply during the first week after exposure in both PBLs and splenocytes. This pattern was followed by a slower decline to baseline over the next week. However, high-frequency cell (HFC) analysis indicates that significant numbers of HFCs persist in the PBLs through day 28 and splenocytes at day 99 post exposure. Mathematical modeling of the time-response curves indicates that the average life span of the majority of AZQ-induced SCE-producing lesions in murine PBLs and splenocytes responsive to phytohemagglutinin is between 3 and 5 days.

Noncirrhotic portal hypertension after arsenic ingestion.

Five years after arsenic therapy, the patient described had noncirrhotic portal hypertension, for which he had splenectomy and anastomosis of the splenic vein to the left renal vein. During the 12-year postoperative period he had Bowen's disease (skin carcinoma), but has had normal liver function and no further gastrointestinal bleeding. Arsenic exposure in humans is common throughout the world and may lead to late complications such as noncirrhotic portal hypertension and skin carcinoma, as well as malignancies of the lungs, liver, and lymphatic systems. It may also lead to severe arteriosclerosis with involvement of the heart and extremities.

Complications associated with carotid endarterectomy.

A discussion of some of the complications associated with carotid endarterectomy is presented. Accurate knowledge of anatomy is essential in preventing injury to nerves, blood vessels and other regional anatomical structures. Complications following carotid artery operation may be severe and must be avoided if possible. Indications for carotid endarterectomy are still controversial. The final decision for or against operation rests on the judgment of the vascular surgeon.

The nature of arterial healing following dextran treatment of experimental small artery trauma.

Vascular thrombosis is a major cause of morbidity and death. Because of the many variables involved with thrombosis in patients, major advances in treatment often depend upon design and study of adequate experimental models which provide a degree of control of the variables. Arterial trauma was produced in small femoral arteries 3 mm. or less in diameter by a standardized intimectomy technique. One group of animals was treated with an equal volume of saline and served as controls. Serial sections of blood vessels at graded time intervals from one hour to 90 days were studied. The damaged blood vessels of dextran-treated animals did not thrombose and provided an opportunity for studying the mechanism of healing in traumatized blood vessels which remained patent. The damaged blood vessels of saline-treated animals uniformly thrombosed and eventually healed for scar formation with evidences of attempts at recanalization. The blood vessels of dextran-treated animals remained open for as long as 90 days and were re-endothelialized and healed. What appears to be beginning re-endothelialization of blood vessels of dextran-treated animals was observed as early as 48 hours. In a model experimental setting, dextran has been shown to prevent thrombosis and permit healing in small arteries subjected to a standardized surgical trauma.