Integration of sperm DNA damage assessment into OECD test guidelines for genotoxicity testing using the MutaMouse model.

The Organisation for Economic Co-operation and Development (OECD) endorses test guidelines (TG) for identifying chemicals that are genotoxic, such as the transgenic rodent gene mutation assay (TG 488). Current OECD TG do not include assays for sperm DNA damage resulting in a critical testing gap. We evaluated the performance of the Sperm Chromatin Structure Assay (SCSA) and the Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick end Labeling (TUNEL) assay to detect sperm DNA damage within the recommended TG 488 protocol. MutaMouse males received 0, 0.5, 1, or 2 mg/kg/day triethylenemelamine (TEM), a multifunctional alkylating agent, for 28 days orally and tissues were collected two (blood) and three (sperm and bone marrow) days later. TEM significantly increased the frequency of lacZ mutants in bone marrow, and of micronuclei (MN) in both reticulocytes (%MN-RET) and normochromatic erythrocytes (%MN-NCE) in a dose-dependent manner (P < 0.05). The percentage of DNA fragmentation index (%DFI) and %TUNEL positive cells demonstrated dose-related increases in sperm (P < 0.05), and the two assay results were strongly correlated (R = 0.9298). Within the same animal, a good correlation was observed between %MN-NCE and %DFI (R = 0.7189). Finally, benchmark dose modelling (BMD) showed comparable BMD10 values among the somatic and germ cell assays. Our results suggest that sperm DNA damage assays can be easily integrated into standard OECD designs investigating genotoxicity in somatic tissues to provide key information on whether a chemical is genotoxic in germ cells and impact its risk assessment.

Quantitative dose-response curves from subcellular lipid multilayer microarrays.

The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate.

Triethylenemelamine: an initiator of two-stage carcinogenesis in mouse skin which lacks the potential of a complete carcinogen.

The dorsal skin of female CD-1 mice was treated with triethylenemelamine (TEM) to determine whether this agent acted either as a complete carcinogen or as an initiator of carcinogenesis. A dose of 0.01-1.0 mumol of TEM applied once a week for 32 weeks to the skin of the backs of mice did not produce any detectable tumors. A dose of 2.5 mumol applied once a week over the same period produced only a single papilloma in a group of 20 mice. However, when mice were treated with a single dose of 1 mumol of TEM followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week, 88% of the mice produced papillomas after 28 weeks. Using the same protocol, a single application of hexamethylmelamine (HMM), pentamethylmelamine (PMM), or melamine followed by promotion with TPA had no significant tumor initiating activity. These data suggest that TEM acts primarily as an initiator of two-stage carcinogenesis.

Initiating carcinogen, triethylenemelamine, induces micronuclei in skin target cells.

Keratinocytes from mouse skin were cultured for a short period in vitro following single or multiple treatments at low dose levels in vivo with the known chromosome-damaging agent triethylenemelamine (TEM). The chemical was applied to the skin of HRA/Skh hairless mice at concentrations corresponding to those reported to initiate cancer in initiation-promotion assays. A significant dose-related depression in keratinocyte cell recovery occurred over the dose range 0.3-1 mg TEM/mouse (single or multiple treatments). Under the same conditions, a dose-related induction of micronuclei was observed using the cytokinesis-block method with cytochalasin B. A similar frequency of micronuclei was detected in binucleate cells from mice treated with single or multiple applications of TEM. Mice held for 12-48 h post-treatment, before removal of skin for in vitro culture, yielded highest micronuclei frequencies. These results indicate that the same target cell population, skin keratinocytes, can be used to investigate both genotoxicity and carcinogenesis, and that micronucleus induction in these cells may be a sensitive signal of skin cancer initiation.

Detection of TEM-induced reciprocal translocations in F1 sons of CD-1 male mice: comparison of sequential fertility evaluation and cytogenetic analysis.

To determine the positive and negative classification error rates associated with the HTA in our laboratory, F1 sons of TEM-exposed CD-1 male mice were evaluated by the sequential fertility method with subsequent cytogenetic analysis. Males who sired three litters of size 10 or less when mated to primiparous females from either the B6C3F1 or the BCF1 strain were classified as partial steriles. When meiotic chromosome analyses revealed the presence of at least two cells containing multivalent figures, males were classified as translocation heterozygotes. When the fertility evaluation and the cytogenetic analysis were compared, normal fertility was observed on 5 of 83 (6.02%) translocation-bearing F1 males mated to B6C3F1 tester females and on 3 of 83 (3.61%) F1 males mated to BCF1 tester females. Thus, the false-negative error rates were 6.02% and 3.61% with these two tester strains. Multivalent figures were not observed in the meiotic chromosomes of 410 F1 males. Of these, 12 (2.93%) had reduced fertility when mated to the B6C3F1 tester strain as did 7 (1.71%) mated to the BCF1 strain. Thus, the false-positive error rates with these two tester strains were 2.93% for the B6C3F1 strain and 1.71% for the BCF1 strain. Our results indicate that non-zero error rates, both false-positive and false-negative, are associated with the sequential mating method HTA. In addition, the magnitude of these error rates was influenced not only by the tester female strain but also by the genotype of the F1 male.

Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry.

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, and 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest (1.0 mg/kg daily x 5) dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated (.87-.93, P less than .001) with dose and with each other. Data obtained from the sperm chromatin structure essay (SCSA) on fresh sperm was highly correlated with measurements of aliquots of the same sample collected over 44 wk, frozen, and then measured on the same day. Sperm head morphology and sperm chromatin structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

Dose- and time-response relationships of triethylenemelamine-induced chromosomal aberrations in rat bone marrow cells.

Groups of 18 male Sprague-Dawley rats were administered single i.p. doses of 0.00, 0.125, 0.25 or 0.50 mg/kg of triethylenemelamine (TEM). 6 rats per treatment group were killed 6, 24 or 48 h after dosing, and bone marrow cells were collected and prepared for cytogenetic analysis. Under the present conditions, 0.25 mg/kg with sampling of bone marrow cells 24 h after treatment appeared to represent optimal conditions for using TEM as a positive control agent in the rat in vivo cytogenetic assay as described.

The impact of severely damaged cells in cytogenetic research.

In a large multilaboratory cytogenetic study of interlaboratory variations using 4 dose levels of triethylenemelamine and a control, a severely damaged cell was operationally defined as a cell which contained at least 10 aberrations of any type. A review of this study suggested that the use of this definition introduced a bias in the measurement and interpretation of results for the other cytogenetic categories studied. As a result, the original severely damaged cells were carefully reanalyzed to investigate the characteristics of this bias and to seek procedures to minimize or eliminate it. Results characterize this bias and demonstrate that when a severely damaged cell is defined as one containing at least 20 aberrations and those aberrations in the remaining non-severely damaged cells are classified by specific type, the bias is significantly reduced and chromosome analysis can be improved as a test system.

Dominant lethal test response with IMS and TEM using different combinations of male and female stocks of mice.

We conducted dominant lethal studies with chemical mutagens IMS and TEM to investigate the influence which different treated male stocks might have upon individual female response. In both studies, treated males from a CD-1 random bred stock and each of two F1 hybrid stocks (C57BL/6N X C3H; C57BL/6J X BALB/c) were mated to untreated females from the two F1 hybrid stocks. We found that variation in response due to the treatment effect was modulated by the specific male stock/female stock combination involved. The male-dependent variation observed prevented any meaningful evaluation of relative female stock sensitivity but it was obvious that factors other than differences in repair capacity of female germ cells contributed to the response differences observed.

Heat-shocks prior to treatment of Vicia faba root-tip meristems with maleic hydrazide or TEM reduce the yield of chromatid aberrations.

Heat-shocks (10 and 30 min at 40 degrees C) prior to treatment with MH or TEM significantly reduced the yield of metaphases with chromatid aberrations. No such effect was observed when ethanol was used for aberration induction. The 'heat-shock effect' on aberration induction by MH and TEM is comparable to 'clastogenic adaptation' observed after pretreatment ('conditioning') with low clastogen concentrations prior to 'challenging' with high clastogen concentrations; both require unimpaired protein synthesis.

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides with triethylenemelamine.

A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.

Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Vicia faba root tip meristem cells.

When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of Vicia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24 degrees C to 12 degrees C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.

Sister-chromatid exchanges in Picea abies--a test for genotoxicity in forest trees.

A genotoxicity test, based on the evaluation of sister-chromatid exchange frequencies, has been developed for the spruce fir. The basic frequency was 36.9 SCEs/cell. Mitomycin C treatment (MMC, 5 x 10(-6) M, 0.5 h) doubled the 'spontaneous' SCE frequency, maleic hydrazide treatment (MH, 5 x 10(-4) M, 0.5 h) increased it nearly 7-fold. This corresponds to data obtained previously for Vicia faba. Chromatid-type aberrations were induced by the same mutagens (MH, 5 x 10(-4) M, 0.5 h; MMC 2 x 10(-5) M, 1 h) or by triethylenemelamine (TEN, 2 x 10(-4) M, 0.5 h). MH treatment resulted in aberration yields comparable to those observed in Vicia faba, MMC and TEM were less efficient aberration inducers in P. abies. While SCEs may be counted for single chromosomes, for reliable evaluation of chromatid aberrations large numbers of complete and well spread metaphases have to be inspected.

Premature chromosome condensation, structural chromosome aberrations, and micronuclei in early mouse embryos after treatment of paternal postmeiotic germ cells with triethylenemelamine: possible mechanisms for chemically induced dominant-lethal mutatiions.

Cytogenetic effects in preimplantation 4-8-cell mouse embryos have been investigated after treating paternal postmeiotic germ cells with triethylenemelamine (TEM). Dose-levels of TEM which do not affect fertilization but yield high incidence of dominant-lethal mutations in sperm and spermatids were shown to produce relatively high frequencies of (a) premature chromosome condensation (PCC), (b) structural chromosome anomalies (breakage-reunion phenomena), and (c) micronuclei in these embryos. The results indicate that genetic death of embryos is mainly due to imbalance (i.e. loss) of genetic material, either from breaks leading to lagging fragments and micronuclei, or from the segregation of various types of exchange figures (dicentrics, rings etc.) resulting in mechanical disturbances of cleavage division. It is suggested that PCC, to some extent, is an expression of TEM-induced long-lived lesions which, transmitted into the egg, prevent the chromosomes in question from replicating and/or condensing normally. This phenomenon could well be associated with loss of chromosome material resulting in embryonic death.

Involvement of phytochelatins in NiCl2-triggered protection against induction of chromatid aberrations by TEM and MH in Vicia faba root tip meristems?

Conditioning treatment of Vicia faba root tip meristem cells with NiCl2 prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) triggered protective functions against both these clastogens, i.e., resulted in a significantly reduced yield of metaphases with chromatid aberrations. Protection was prevented by pretreatment with buthionine sulfoximine (BSI), an inhibitor of the synthesis of plant phytochelatins (PCs), indicating that the NiCl2-triggered PC synthesis may be involved in the protective functions induced by NiCl2 conditioning treatment. BSI (instead of NiCl2) conditioning treatment triggered protection against MH but not against TEM.

Combined application of MH and TEM for conditioning treatment simultaneously triggers two protective functions against induction of chromatid aberrations in Vicia faba root tip meristem cells.

Conditioning pre-exposure of Vicia faba root tip meristem cells to triethylenemelamine (TEM) does not trigger an adaptive response to maleic hydrazide (MH) and vice versa. Since TEM conditioning treatment can induce protective effects (as evident from the yield of metaphases with chromatid aberrations) against TEM challenging treatment and MH conditioning can trigger an adaptive response to MH challenging treatment, two different protective functions are apparently triggered in dependence on the agent used for conditioning pre-exposure. When a mixture of TEM and MH is being used for conditioning treatment, adaptive responses to both TEM and MH can simultaneously be induced and significantly reduce the yield of metaphases with chromatid aberrations observed after challenge treatment with TEM or MH.

Chromosome analysis of human spermatozoa exposed to antineoplastic agents in vitro.

We studied in vitro the cytogenetic effects of six antineoplastic agents, bleomycin (BM), cyclophosphamide (CP), daunomycin (DM), methyl methanesulfonate (MMS), mitomycin C (MMC) and triethylenemelamine (TEM) on spermatozoa, using an interspecific in vitro fertilization system between zona-free hamster oocytes and human or bull spermatozoa. In preliminary experiments with bull spermatozoa, clastogenic effects were clearly shown with BM, DM, MMS and TEM, but not with CP and MMC. In main experiments, the effects of the first four chemicals were studied in detail with human spermatozoa. Total numbers of 585 and 512 spermatozoa were karyotyped in the control and the chemical-treated groups respectively. The incidence of spermatozoa with structural chromosome aberrations was 34.5%, 53.0%, 59.3%, and 55.6% in the BM (50 micrograms/ml, 90 min), DM (0.1 microgram/ml, 90 min), MMS (100 micrograms/ml, 120 min) and TEM (0.1 micrograms/ml, 120 min) groups respectively, each showing a significantly higher incidence than the matched controls (10.1-13.5%). Breakage-type aberrations were more frequent than exchange-type aberrations in the BM, MMS and TEM groups, while the exchange-type aberrations were more frequent in the DM group. Exchanges were mainly of the chromatid type in the DM, MMS and TEM groups, while chromosome-type exchanges occurred more frequently in the BM group. These results are discussed in relation to previous data on chemical-induced chromosome aberrations in mammalian somatic cells and in mouse spermatozoa.

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays.

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.

Genotoxic potency in mouse spermatogonial stem cells of triethylenemelamine, mitomycin C, ethylnitrosourea, procarbazine, and propyl methanesulfonate as measured by F1 congenital defects.

Male ICR mice were intraperitoneally injected with TEM, MMC, ENU, PCZ, or PMS and mated to untreated females of the same strain on days 64-80 after the treatment. Copulations during this period involve sperm that were spermatogonial stem cells at the time of the treatment. The fetuses were examined on day 18 of pregnancy for external and skeletal abnormalities. The 5 mutagens tested all caused significant increases in the incidence of abnormal fetuses over the control level. The genotoxically effective dose, in mmole/kg, for producing fetal abnormalities with a frequency of 2% was estimated to be 0.007 for TEM and MMC, 0.6 for ENU, 1.8 for PCZ, and 3.0 for PMS. These values correlate well with the mutagenic potency estimated from the data reported for inducing specific-locus mutations in spermatogonial stem cells. Irrespective of the kind of mutagen used, external abnormalities represented by cleft palate and dwarfism occurred more frequently than skeletal abnormalities represented by rib malformations. It is concluded from these data that F1 fetal abnormalities can serve as sensitive indicators for quantitatively assessing the genotoxicity of a chemical agent in spermatogonial stem cells.

Triethylene melamine-induced sister-chromatid exchange in murine lymphocytes exposed in vivo.

The induction of sister-chromatid exchange (SCE) by triethylene melamine (TEM), a known animal carcinogen, was investigated in an in vivo exposure/in vitro culture murine lymphocyte assay. Dose-related increases in SCE were observed in B6D2F1 mice following a single i.p. injection of 0.5, 1 or 2 mg/kg TEM. SCE frequencies remained elevated over baseline levels at 24 h post exposure. It is hoped that studies of this nature can determine whether the in vivo/in vitro murine lymphocyte SCE assay is useful for predicting the carcinogenic potential of an agent.