[Chronic, refractory ulcer on the ring finger : Manifestation of distal epithelioid sarcoma]. / Chronisches, therapieresistentes Ulkus am Ringfinger : Manifestation des epitheloiden Sarkoms vom distalen Typ.

The epithelioid sarcoma classic, "distal" type was first published in 1970. It is a very rare, malignant, aggressive subcutaneous soft tissue sarcoma that shows characteristic positivity for both epithelial and mesenchymal immunohistochemical markers. It grows very slowly and mostly presents in young men. Clinically the tumor is characterized as a coarse cutaneous or subcutaneous nodular induration that often ulcerates in the course of the disease. An association with trauma is often described and can lengthen time to diagnosis. Most frequently it is found on the flexural side of fingers, the back of the hands, soles of the feet, and extensor sides of arms and legs. Specific for this type of sarcoma is the progression along nerves, tendons, and fasciae. Treatment of choice should be wide excision of the tumor, sentinel node biopsy, and possibly even localized postoperative radiation therapy. Unfortunately the epithelioid sarcoma is very likely to recur and is then associated with metastases in the lung and lymph nodes.

Dose-response relationship of temozolomide, determined by the Pig-a, comet, and micronucleus assay.

Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RETCD59-) and at 3.75 mg/kg/day for mutant red blood cells (RBCCD59-), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.

Diagnostically challenging human papillomavirus-associated primary squamous cell carcinoma of the rectum with metastasis in both ovaries: a case report.

INTRODUCTION: Squamous cell carcinomas of the rectum are extremely rare and their pathogenesis is still under debate. Their proper diagnosis and treatment may thus be challenging. CASE PRESENTATION: A 52-year-old Caucasian woman was transferred to our department with a history of pelvic pain. Colonoscopy revealed a small tumorous lesion of the upper rectum and an endoscopic biopsy showed infiltration of the rectal mucosa by a squamous cell carcinoma. Afterward, tumorous lesions were found on imaging in both her ovaries. A laparoscopy with adnexectomy and anal mapping was performed and revealed tumor masses of squamous cell carcinoma in both ovaries. Based on the large size of the ovarian tumors and the concurrence of extensive, partly ciliated, macrocystic epithelium in one of the ovaries, a diagnosis of ovarian squamous cell carcinoma arising from a mature teratoma was rendered. However, human papillomavirus genotyping analyses were positive for human papillomavirus-16 in both the rectal tumor and ovarian tumors leading to a final diagnosis of a human papillomavirus-associated rectal squamous cell carcinoma metastatic to both ovaries. Neoadjuvant chemoradiation therapy of her rectum, total mesorectal excision, and hysterectomy were performed followed by adjuvant chemotherapy. CONCLUSION: Colorectal squamous cell carcinoma is a rare disease. In cases of colorectal squamous cell carcinoma, metastatic disease at any other location has to be excluded. Human papillomavirus genotyping is essential in this context. Discussion of the treatment strategies should be interdisciplinary and include chemoradiation therapy and radical surgery.

Analysis of historical negative control group data from the rat in vivo micronucleus assay.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Placental involvement in glycogen storage disease type IV.

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by glycogen branching enzyme (GBE) deficiency and resulting in the storage of abnormal glycogen (polyglucosan). Prenatal diagnosis is based on biochemical assay of GBE activity or on mutation analysis, but polyglucosan can also be identified histologically in fetal tissues. We document placental involvement at 25 and 35 weeks of gestation in two cases with genetically confirmed GSD IV. Intracellular inclusions were seen mainly in the extravillous trophoblast. Our findings suggest the possibility of prenatal diagnosis by histological evaluation of placental biopsies.

Differential micronucleus frequency in isogenic human cells deficient in DNA repair pathways is a valuable indicator for evaluating genotoxic agents and their genotoxic mechanisms.

The micronucleus (MN) test has become an attractive tool both for evaluating the genotoxicity of test chemicals because of its ability to detect clastogenic and aneugenic events and for its convenience. As the MN assay has been mostly performed using only DNA repair-proficient mammalian cells, we believed that the comparison of the MN frequency between DNA repair-proficient and -deficient human cells may be an excellent indicator for detecting the genotoxic potential of test chemicals and for understanding their mode of action. To address this issue, the following five genes encoding DNA-damage-response (DDR) factors were disrupted in the TK6 B cell line, a human cell line widely used for the MN test: FANCD2, DNA polymerase ζ (REV3), XRCC1, RAD54, and/or LIG4. Using these isogenic TK6 cell lines, the MN test was conducted for four widely-used DNA-damaging agents: methyl methanesulfonate (MMS), hydrogen peroxide (H2 O2 ), γ-rays, and mitomycin C (MMC). The frequency of micronuclei in the double strand break repair-deficient RAD54-/- /LIG4-/- cells after exposure to γ-rays, H2 O2 , MMS and MMC was 6.2-7.5 times higher than that of parental wild-type TK6 cells. The percentages of cells exhibiting micronuclei in the base excision repair- and single strand break repair-deficient XRCC1-/- cells after exposure to H2 O2 , MMC and MMS were all ∼5 times higher than those of wild-type cells. In summary, a supplementary MN assay using the combination of RAD54-/- /LIG4-/- , XRCC1-/- and wild-type TK6 cells is a promising method for detecting the genotoxic potential of test chemicals and their mode of action. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.

Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.

beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.

Effect of 3'-methoxy-4'-nitroflavone on benzo[a]pyrene toxicity. Aryl hydrocarbon receptor-dependent and -independent mechanisms.

This laboratory has studied a number of flavone derivatives for aryl hydrocarbon receptor (AhR) agonist and antagonist potential using cell-free and cell culture systems. The current report extends these investigations by testing the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) for in vivo activity. Wild-type C57Bl/6 male mice were treated with solvent, benzo[a]pyrene (B[a]P; 150 mg/kg), or concurrently with B[a]P and 3'M4'NF (60 mg/kg; delivered as a split dose). Since B[a]P is bioactivated to genotoxic metabolites by AhR-regulated enzymes, we measured B[a]P-induced chromosomal damage in peripheral blood (i.e. micronuclei) to characterize the antagonistic potential of 3'M4'NF in vivo. The influence of AhR signal transduction was investigated further by challenging wild-type and Ahr null allele mice with B[a]P with and without a 3'M4'NF co-treatment. The micronucleus data obtained from these experiments indicated that 3'M4'NF can attenuate the genotoxicity of B[a]P significantly. Since 3'M4'NF also protected Ahr null allele mice from B[a]P-induced genetic damage, it was apparent that AhR-independent mechanisms contribute to the effects observed. However, as opposed to the protective effects observed with the micronucleus endpoint, histological observations and lethality data indicated that some B[a]P effects are enhanced by 3'M4'NF. Potentiated B[a]P toxicity may be explained by inhibition of basal and induced CYP1A1/2 activities. Both in vitro and in vivo data presented herein support this hypothesis.

Chondrocytic cell differentiation in clear cell chondrosarcoma.

Clear cell chondrosarcoma is a rare mesenchymal neoplasm of unclear differentiation. Besides having a chondrogenic nature, an osteogenic differentiation was also proposed. In this study, expression analysis of extracellular matrix genes, which are specific for different mesenchymal cell differentiation pathways, were used to get a better understanding of origin and differentiation pattern of the clear cell chondrosarcoma tumor cells. Our in situ analysis of two cases shows that (1) chondrocytic cell differentiation as marked by the expression of cartilage collagen type II and proteoglycans is a characteristic feature within the development of the neoplasm, (2) multifocal chondrocyte hypertrophy as shown by the expression of type X collagen does occur, and (3) no significant expression of collagen type I, the main gene product of osteoblastic cells, is found by the neoplastic cells. Thus, our study indicates that clear cell chondrosarcoma shows a chondrogenic, but not osteogenic, differentiation and represents a true chondrosarcoma. The unusual scarcity of its extracellular and the multifocal expression of type X collagen marks clear cell chondrosarcoma as a chondrosarcoma tumor entity of a particular cell differentiation pattern. The expression of cartilage type collagens represents a distinct marker from bone metastases of clear cell neoplasms of other origins.

In vivo antagonism of AhR-mediated gene induction by 3'-methoxy-4'-nitroflavone in TCDD-responsive lacZ mice.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a member of the bHLH-PAS family of proteins. The highest-affinity ligand of this receptor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent immunological, reproductive, and developmental toxicant. The mechanism of TCDD-induced toxicity and the gene modulations that result in toxicity have not been fully defined. The majority of work to date exploring AhR function has focused on agonist-activated AhR signaling. However, it is expected that a better understanding of AhR antagonism will lead to an improved understanding of TCDD toxicity and other AhR-mediated events. This study contributes to such investigations by utilizing the AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) and a dioxin-responsive lacZ transgenic mouse model to characterize antagonism of the receptor system in vivo. The dose-response and time course of TCDD-induced transgene activation were evaluated in transgenic mice to provide information necessary to design 3'M4'NF in vivo studies. TCDD induction of the transgene was noted as early as 8 h after exposure in the lung. 3-miccrog/kg body weight TCDD was the lowest dose found to induce the reporter transgene. Finally, experiments were performed to evaluate the in vivo efficacy of 3'M4'NF. We found that 3'M4'NF inhibits TCDD-mediated reporter gene activation and CYP1A1 induction in vivo. Based on these findings, it is clear that DRE-lacZ animals and the antagonist 3'M4'NF represent important tools which will help in the identification of tissues where AhR is active, and to further characterize AhR-mediated signaling.

In vitro system for detecting non-genotoxic carcinogens.

Chemical risk assessment has been limited by the inability of in vitro short-term assays to identify the true carcinogenic potential of many substances. Numerous methods exist for identifying mutagenic and clastogenic agents, but a practical means of identifying non-genotoxic carcinogens has remained elusive. Experiments described here suggest that some chemicals may participate in carcinogenesis by modulating the enzymatic processes of drug metabolism. The tumor promoters butylated hydroxyanisole, butylated hydroxytoluene, deoxycholic acid, reserpine, trypan blue, and 12,-O-tetradecanoyl phorbol-13-acetate were chosen as model non-genotoxic carcinogens. The enzyme-modulating action of these chemicals was measured using a modified Ames plate incorporation assay whereby the known tumor promoters were plated with a promutagen in the presence of a mammalian metabolic activation system (S9). Each of the non-genotoxic carcinogens significantly increased the mutagenic response of metabolically activated promutagen(s). These experiments suggest that the carcinogenic role of some chemicals may be attributed to their ability to modify the biochemical pathways of drug metabolism. By enhancing or inhibiting the activity of various enzymes, some tumor promoters may create an environment that increases a cell's mutational burden, thereby contributing to neoplastic transformation.

An automated method for discriminating aneugen- vs. clastogen-induced micronuclei.

A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between aneugen- and clastogen-induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC-conjugated anti-CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single-parameter histogram. The histogram statistic "median channel" was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or aneugenic activity, and 2) MN resulting from aneugens can be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Modulation of phorbol ester-induced HL-60 differentiation by prostaglandin E2.

When treated with phorbol tumor promoters, HL-60 cells undergo terminal differentiation evidenced by a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity. Internalization of fluorescent particles by cells exhibiting the phagocytic positive phenotype (phag+) provides a sensitive indication of promoter-induced differentiation, and the resulting fluorescent cells can be quantitatively analyzed by flow cytometry. The current study was initiated to further test the predictive power of a flow cytometry based HL-60 differentiation assay in the detection of agents associated with tumor promotion. Specifically, experiments were designed to assess the sensitivity of the test system to co-promoters which enhance promoter activity in vivo. Prostaglandin E2 (PGE2) was chosen as a model co-promoter since it has been shown to potentiate phorbol ester (i.e. 12-O-tetradecanoyl phorbol-13-acetate; TPA) induced biological effects in vivo. Results detailed in the current report indicate that PGE2 enhances TPA-induced differentiation of HL-60 cells in a dose-dependent manner. As with in vivo co-promotion experiments, PGE2 exhibited a maximum potentiating effect when administered prior to TPA. These data indicate that HL-60 cells are not only sensitive to phorbol promoters, but also to the co-promoter PGE2. These experiments support the hypothesis that a flow cytometry based HL-60 assay may prove useful for studying chemical agents or intrinsic cellular factors that are involved in the tumor promotion phase of carcinogenesis.

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow.

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10,000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.

Development of a sensitive in vitro method for identifying tumor promoters.

The identification and characterization of nongenotoxic carcinogens represents a significant challenge to toxicologists. In vitro methods for identifying tumor promoters with suitable sensitivity and specificity have been particularly elusive. Experiments are described which suggest that the human promyelocytic leukemia cell line HL-60 provides a sensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture which exhibits high phagocytic activity. Fluorescent latex particles were used as sensors to highlight the phagocytic phenotype and permitted the use of flow cytometry to automatically quantitate particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tumor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo promoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 pg of TPA per ml caused statistically significant increases in phagocytic activity. The experiments described herein suggest that in vitro differentiation of HL-60 cells may reflect the promoter-dependent modifications to gene expression that are observed in vivo during the promotion phase of carcinogenesis. The described method may represent a sensitive promoter screening assay which is both rapid and economical.

Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood.

Micronucleated cells (MN cells) generated spontaneously or by clastogen action accumulate in the peripheral blood of the mouse, and their presence reflects the level of chromosome damage. Traditionally, micronucleated cells have been scored by visual inspection. With the development of flow cytometry based scoring procedures, vast numbers of cells can be analyzed, making it possible to determine the change in the number of MN cells in the total peripheral blood pool. This report describes experiments whereby initial blood samples were obtained before dosing, providing mouse-specific controls for measuring subsequent changes in MN cells. Mice were then dosed with saline (solvent control), methyl methanesulfonate, cyclophosphamide or colchicine every 48 h and bled every 96 h for 12 days. For each blood sample, one million fixed erythrocytes (RBCs) were interrogated for the presence of micronuclei, and regression analysis was used to determine the rate of MN cell influx per day for each animal or sets of animals. To evaluate the effect of treatment on MN induction, the mean slopes of solvent and chemically treated animals were compared using t-tests. The results of these experiments indicate that the kinetics of MN induction continues near the background frequency for saline dosed mice, whereas clastogenic agents or spindle poisons cause a significant influx of MN events into the blood. The results suggest that some studies may benefit from a flow cytometry based analysis of multiple blood samples, especially when the number of mice is limited, or when a weak clastogen is being investigated.

Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer.

A flow cytometric procedure for scoring micronuclei in mouse peripheral blood erythrocytes, especially reticulocytes, is described. The methods reported herein were developed in an effort to simplify the techniques and to reduce the equipment requirements associated with automated micronucleus analyses. With this procedure, fluorescein-conjugated monoclonal antibodies which bind to the CD71-defined antigen (the transferrin receptor) are used to label reticulocytes. The nucleic acid dye propidium iodide is used to identify cells with micronuclei. Given 488 nm excitation, four populations of erythrocytes are clearly resolved: normochromatic erythrocytes with and without micronuclei, and reticulocytes with and without micronuclei. Since the method is capable of simultaneously providing the incidence of micronuclei in both mature and immature erythrocyte populations, it is compatible with either chronic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood micronuclei, an experiment with the model clastogen methyl methanesulfonate is described. Additionally, a reconstruction experiment was performed whereby three mouse blood samples were spiked with successively greater volumes of blood from a clastogen-treated animal so each preparation differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cytometry so that the two methods could be directly compared. Collectively, the results from the methyl methanesulfonate experiment and the reconstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughout scoring methodology are clearly indicated.

Induction of micronuclei by low doses of azidothymidine (AZT).

The dideoxynucleoside azidothymidine (AZT; Zidovudine) was assessed for its ability to induce micronuclei in mouse erythrocytes at a low (therapeutic) dosage. Specifically, male and female BALB/c mice were treated via intraperitoneal injection 5 days a week for 2 weeks with saline or 17 mg AZT/kg body weight per day. Each animal was monitored for chemical-induced micronucleus formation over the course of the treatment regimen through the flow cytometric analysis of one million pre-dosing and one million post-dosing peripheral blood erythrocytes. No significant change in micronucleus frequencies was observed for the vehicle control group as micronuclei continued to enter the peripheral blood pool at background levels. Conversely, the AZT-treated mice exhibited a statistically significant net increase in micronucleated cells over the course of dosing as erythrocytes with a high incidence of micronuclei entered the peripheral blood pool. The advantages of high throughput scoring protocols utilizing flow cytometry are discussed.