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This study examines the thermal behavior of a laser ignited thermite composed of aluminum and bismuth trioxide. Temperature data were collected during the reaction using a four-color pyrometer and a high-speed color camera modified for thermography. The two diagnostics were arranged to collect data simultaneously, with similar fields of view and with similar data acquisition rates, so that the two techniques could be directly compared. Results show that at initial and final stages of the reaction, a lower signal-to-noise ratio affects the accuracy of the measured temperatures. Both diagnostics captured the same trends in transient thermal behavior, but the average temperatures measured with thermography were about 750 K higher than those from the pyrometer. This difference was attributed to the lower dynamic range of the thermography camera's image sensor, which was unable to resolve cooler temperatures in the field of view as well as the photomultiplier tube sensors in the pyrometer. Overall, while the camera could not accurately capture the average temperature of a scene, its ability to capture peak temperatures and spatial data make it the preferred method for tracking thermal behavior in thermite reactions.

The impact of laser-driven flyer plates on energetic materials CL-20, PETN, and TATB has been investigated. Flyer plates composed of 25 μm thick Al were impacted into the energetic materials at velocities up to 1.3 km/s. The flyer plates were accelerated by means of an Nd:YAG laser pulse. The laser pulse generates rapidly expanding plasma between the flyer plate foil and the substrate to which it is adhered. As the plasma grows, a section of the metal foil is ejected at high speed, forming the flyer plate. The velocity of the flyer plate was determined using VISAR, time of flight, and high-speed video. The response of the energetic material to impact was determined by light emission recorded by an infrared-sensitive photodiode. Following post-impact analysis of the impacted energetic material, it was hypothesized that the light emitted by the material after impact is not due to the impact of the flyer itself but rather is caused by the decomposition of energetic material ejected (via the shock of flyer plate impact) into a cloud of hot products generated during the launch of the flyer plate. This hypothesis was confirmed through schlieren imaging of a flyer plate launch, clearly showing the ejection of hot gases and particles from the region surrounding the flyer plate launch and the burning of the ejected energetic material particles.

There is a wealth of information in the published literature which describes a multitude of approaches to enriching the environment of laboratory animals. This paper attempts to review the various methods of enrichment through social contact, enhancement of the environment and diet, and improvements in husbandry. It attempts to place the various enrichment initiatives within the context of a laboratory which conducts regulatory toxicology, describes some of the experiences in the author's own laboratory and attempts to highlight those ideas which might prove practical to implement in the future. The aim is to demonstrate that a creative approach to environmental enrichment is indeed compatible with regulatory toxicology. It is hoped that this will encourage those responsible for the care and welfare of animals in such a laboratory to challenge historical practices and include environmental enrichment as a fundamental necessity of study design.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.

Transgenic mouse mutation assays, such as MutaMouse (lacZ, CD2F1) and Big Blue (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both MutaMouse and Big Blue should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics assays in bone marrow or peripheral blood.

Benzo[a]pyrene (BP) has been investigated for the ability to induce mutation at the site of contact. Skin painting treatments with BP caused a time-dependent and statistically significant increase in mutation frequency (MF) in the treated areas of skin. The MF exceeded 500 x 10(-6) 21 days after either 1 x 25 or 5 x 5 micrograms treatments. Increases to > 700 x 10(-6) were seen when doses of 1 x 50 or 5 x 10 micrograms were used. Neither the liver nor the lung showed any increase in mutation frequency after 21 days in animals exposed to the 5 x 10 micrograms treatment regime. It is concluded that following topical administration, BP is able to induce mutation in the skin at the site of application, but not in either the lung or liver.

Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a collaborative study, the effects of the direct-acting mutagens, methyl methanesulphonate (MMS) and ethylnitrosourea (ENU), were investigated for gene mutation in the tubular sperm of Muta Mice testes after a single intraperitoneal exposure. Groups of male Muta Mice were dosed intraperitoneally with either 1/15 M phosphate buffer, pH 6.0 (vehicle control), 40 mg/kg methyl methanesulphonate (MMS) or 150 mg/kg ethylnitrosourea (ENU). The animals were sacrificed 14 days after the single dose. Mutation frequencies were determined in tubular sperm DNA. The results showed a mean mutation frequency (MF) of 2.1 x 10(5) (64 mutants per 3.05 x 10(6) PFU) for the 10 vehicle-treated mice, a mean MF of 2.8 x 10(5) (78 mutants per 2.75 x 10(6) PFU) for the 10 MMS-treated mice and a mean MF of 9.1 x 10(5) (194 mutants per 2.14 x 10(6) PFU) for the 8 ENU-treated mice; this latter value representing a 4.5-fold increase over the vehicle control values.

Transgenic mouse assays, such as MutaMouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a validation study, the effects of the direct-acting mutagens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-chloromethyl-pyrene (CMP), were investigated for gene mutation in the tissues of MutaMice after a single oral or topical exposure. MNNG (50 or 100 mg/kg) or CMP (25 or 50 mg/kg) were administered as a single oral dose and the mice killed after 3, 7 or 10 days. Mutation frequencies were determined in stomach DNA from both MNNG and CMP-treated animals and in liver DNA from the MNNG-treated animals only. The results, although obtained from a limited number of animals, consistently showed that MNNG increased the mutation frequency in stomach DNA, but not apparently in liver DNA, at each exposure time; no clear increase in mutation frequency was seen in the stomach DNA of CMP-treated animals. Also, MNNG (250 or 500 micrograms) or CMP (5 or 10 micrograms) in acetone were applied as a single dose to the shorn skin of mice 7, 14 or 21 days prior to death. A positive control group was similarly given dimethyl-benz[a]anthracene (DMBA, 40 micrograms) and sacrificed after 14 days. Mutation frequencies were determined in the skin DNA extracted from all animals and in the stomach DNA from MNNG-painted animals only. The results, again obtained from a limited number of animals, clearly showed that all test compounds consistently increased the mutation frequency of skin DNA and that these increases were far greater in the DMBA- and MNNG-treated mice than the CMP-treated mice. No apparent increases were seen in the stomach DNA from the MNNG-painted mice.

Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a validation study, 2-acetylaminofluorene was administered as a single oral dose of either 50 or 100 mg/kg. The mice were killed 3, 7, 14, 28, 56 or 112 days after this single treatment. Mutation frequencies were determined in liver DNA. The results showed that a mutagenic response was observed in the mice treated at the highest dose (100 mg/kg) and this increase was expressed from 28 up to 112 days after the single exposure.

There are currently unresolved discussions on two important topics in regulatory genetic toxicology, namely whether or not it is necessary to confirm clearly negative results from in vitro assays in independent experiments, and whether or not the mammalian cell gene mutation test should be part of a core battery of tests. Analysis of in-house data, using full regulatory protocols, suggests that for bacterial mutation tests (144 compounds reviewed) it is impractical to design a single experiment to incorporate all relevant variables and, therefore, confirmation of negative results using modified methodology is desirable. On the other hand, data from TK mutation assays (65 compounds reviewed) and chromosomal aberration tests (94 compounds reviewed) suggest that confirmation of negative results in well-designed mammalian cell studies is not necessary. Analysis of 32 chemicals, each tested in Ames, TK mutation and chromosomal aberration tests, revealed two positives unique to the TK assay and one unique to the chromosomal aberration test. As the TK assay did not show increased susceptibility to false positives (frequency of positives is similar to other in vitro assays) and these two unique positives were clearly observed (> 2-fold increase in mutation frequency at 60-70% relative survival in both cases), they do appear to be 'real' results. Both compounds induced small colony mutants (one also induced 'large'), and yet in vitro chromosomal aberration and in vivo micronucleus tests were negative. The single unique chromosomal aberration positive may be an artefact of high cytotoxicity, and certainly the substance was negative for micronuclei and UDS in vivo, so it might be argued that the chromosomal aberration test is surplus to requirements. Overall, however, it would seem premature to reject either assay at this time, and experience suggests the extra information provided by two mammalian cell tests instead of one is extremely valuable in assessing risk and deciding upon appropriate follow-up tests in vivo.

In a recent 2-year inhalation study with F344 rats and B6C3F1 mice conducted as part of the U.S. National Toxicology Program (NTP, 1989), chloroethane (ECl) at an exposure concentration of 15,000 ppm induced a high incidence of endometrial uterine carcinomas only in female mice but not in rats, leading to the conclusion of "clear evidence of carcinogenicity" for the mouse. In order to elucidate whether a genotoxic effect may be a critical factor for the carcinogenicity of ECl in the mouse, we have performed three genotoxicity tests: (1) in vitro HPRT test with CHO cells according to a specially developed gas protocol, (2) in vivo/in vitro UDS with female B6C3F1 mice at an exposure concentration of 25,000 ppm (6 h/day, 3 days), (3) in vivo micronucleus assay with male and female B6C3F1 mice exposed to 25,000 ppm ECl according to the same schedule. In the in vitro HPRT test a mutagenic potential of ECl was detected in the presence as well as in the absence of S9 mix. In contrast, both in vivo test systems failed to detect any indications of genotoxicity of chloroethane at an exposure concentration even higher than that of the NTP study. It is suggested that in vivo the genotoxic potential of ECl is so low that an assumed genotoxic damage is below the detection limit of the test systems used. This leads to the conclusion that genotoxicity may not be a key factor in the induction of the uterine carcinomas in the B6C3F1 mouse.

The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved. We describe here a new positive selection protocol which is based upon a modified E. coli bacterial host. This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods.

Three sunscreen ingredients, derivatives of benzylidene camphor, were tested for photomutagenic potential. These were benzenesulfonic acid, 4-[(4,7,7,-trimethyl-3-oxo-bicyclo [2.2.1] hept-2-ylidene) methyl] (Mexoryl SL), 4-(2-oxo 3-bornylidenemethyl) phenyl trimethylammonium methyl sulphate (Mexoryl SO) and 3,3'-(1,4-phenylenedimethylidyne) bis [7,7-dimethyl-2-oxo-bicyclo [2.2.1] heptane-1-methanesulfonic acid] (Mexoryl SX). Two complementary assay systems were used, one involving the induction of reverse mutations in Escherichia coli strain WP2, the other measuring the induction of chromosome damage in Chinese hamster ovary (CHO) cells. Irradiation with UVA and/or UVB was provided by an Osram Ultra-Vitalux sunlamp. None of the three sunscreens, tested either to the limit of solubility or toxicity, gave any indication of photomutagenicity in either assay, under conditions in which the positive control compound, 8-methoxypsoralen, was extremely photomutagenic. It is concluded that Mexoryls SL, SO and SX can be exposed to UV light without producing photomutagenicity measurable using a bacterial reverse mutation or a mammalian chromosome aberration assay.

A new mathematical model has been proposed based on a model presented by Suga, van Dedem, and Moo-Young. The model requires a separate differential equation for each polymeric species (differentiated by degree of polymerization) in the reaction mixture. The main contribution of this model is the incorporation of experimental molecular weight distributions as the initial conditions. These molecular weight distributional as the initial conditions were obtained using modern analytical equipment previouly unknown for this application. The equipment, SEC/LALLS, measures relative concentrations of specific molecular weight species along with the corresponding molecular weights, thus yielding (through some mathematical manipulation) the absolute concentration of each molecular weight species. The concentration at each molecular weight can then be incorporated as the initial condition for that equation. Theoretically, the system of differential equations can be solved to give a more realistic time course of reaction.Synergism between endo-acting and exo-acting enzymes was examined theoretically using the mathematical model. Through model predictions, it was found that synergy is based on two fundamental parameters: (1) each enzyme's activity relative to the sum of enzyme activities and, (2) overall substrate concentration relative to the exo-acting enzyme's Michaeiis kinetic constant K(m). Theoretically, synergism increases as a function of reaction time. Intermediate endo fractions (ratio of endo-acting enzyme activity to the sum of endo-acting and exo-acting enzyme activity) from 0.3 to 0.7 exhibit the most synergism. Values of kappa [log(K(m, exo)/S(0))] above about zero also exhibits the most synergism.An examination of experimental data obtained both by SEC/LALLS and by reducing sugar measurements shows that the model is inadequate for successfully predicting quantities associated with the substrate during reaction. This is especially true for synergism predictions. At short reaction times, the model predicts the data fairly well, but at longer times the predictions are inconsistent with experimental data. These inconsistencies may be due to complicating phenomena such as enzyme inhibitions.

Two complementary assay systems have been adapted for the detection of compounds which may form mutagenic photoproducts during exposure to UV light from an Osram Ultra-Vitalux sunlamp as used in the evaluation of the effectiveness of sun filters. The effects of UVA and of UVB were evaluated. A bacterial plate test using Escherichia coli strain WP2 allowed the bacteria, co-plated with test chemical in soft agar, to be irradiated with various doses of UV light. Mutagenesis was assessed by scoring numbers of tryptophan-independent colonies. The chosen reference compound was 8-methoxypsoralen (8-MOP) which was non-mutagenic alone at the highest dose tested (1000 micrograms/plate). Following simultaneous exposure of bacteria to 8-MOP and doses of UV light which alone had little effect, large numbers of revertants were scored. Numbers of mutants were dependent upon the doses of both 8-MOP and of UV light. The second test system involved the exposure of Chinese hamster ovary cells to UV light in the presence of test chemical to determine the clastogenic effects of photoproducts. Treatment with 8-MOP alone up to 50 micrograms/ml was not clastogenic but concomitant exposure to non-damaging doses of UV light caused large increases in the incidence of chromosome aberrations of all types. Damage was again dependent on the doses of both components. Two additional photoactive compounds, para-aminobenzoic acid and chlorpromazine both show photoclastogenic but not photomutagenic properties. These two complementary assay systems take advantage of using no-effect levels of UV light as a baseline against which photomutagenicity readily can be compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell survival has been measured in normal and Fanconi's anaemia (FA) human fibroblasts after treatment with the bifunctional alkylating agent, nitrogen mustard (HN2). Two FA cell lines exhibited 6- to 10-fold greater sensitivity than the normal cell line. Flow cytometry was used to investigate the effects of HN2 on cell cycle progression of normal and FA cells. After 0.1 microgram/ml HN2 (surviving fraction, s.f. = 0.8) normal cells exhibited an S phase accumulation within 6 h, followed by a transient G2 delay. At higher doses of HN2, the S phase delay became more pronounced and there was considerably greater accumulation of cells in G2. HN2 at 0.01 microgram/ml (s.f. = 0.8) induced no detectable S or G2 delay in FA cells. A higher dose, 0.1 microgram/ml (s.f. = 0.13 and 0.29), again induced no S phase delay, but a gradual accumulation of cells in G2 was observed up to 78 h after treatment. The presence of an S phase delay in normal cells after HN2 treatment may be important in allowing time for DNA repair before completion of DNA synthesis. The absence of such a delay in FA cells suggests that an inability to delay S phase traverse in response to DNA damage from bifunctional alkylating agents may contribute to the sensitivity of FA cells to such drugs.

Cell killing, DNA-interstrand crosslinks, and DNA-protein crosslinks were assayed in nitrogen mustard-resistant Walker 256 carcinoma (WR) cells and the parent cell line (WS) after treatment with 5-[3-(2-chloroethyl)-1-triazenyl]imidazo-4-carboxamide (MCTIC). The WR cells, which also express collateral sensitivity to chloroethylnitrosoureas, were approximately twice as sensitive to the cytotoxic effects of MCTIC as were WS cells. Following treatment with 100 microM MCTIC, there was a rapid accumulation of both DNA-interstrand and DNA-protein crosslinks in the WR cell line, which reached a maximum at 6 and 12 hr, respectively. There was considerably less crosslinking in the WS cells and both cell lines were proficient in repairing most of the crosslinks by 24 hr. Measurement of guanine-O6-alkyl transferase activity showed the enzyme to be present in WS but not in WR cells. These data indicate that the collateral sensitivity of nitrogen mustard-resistant WR cells to chloroethylating drugs is in part due to the loss of guanine-O6-alkyl transferase activity which is present in the parent line.

Two cloned human lymphoblastoid cell lines, Raji and TK6, differ in their sensitivity to the cytotoxic effects of nitrogen mustard (HN2). Raji cells exhibit a biphasic response with an initial D value of 0.06 microgram/ml and a final slope of 0.25 microgram/ml. TK6 cells were considerably more sensitive, D0 value 0.02 microgram/ml. Dose-response relationships for delay in cell cycle progression were measured using flow cytometry. Delay in S-phase traverse was concentration-dependent in both cell lines, and at a given concentration was 2-fold greater in TK6 than in Raji. Numbers of crosslinks (determined by alkaline elution) increased linearly with increasing HN2 concentration and were approximately 2-fold higher in TK6 than in Raji. At equal levels of DNA crosslinks, rates of removal were similar in both cell lines. Inhibition of [3H]TdR uptake was concentration-dependent and the extent of inhibition was similar in both cell lines. Recovery from HN2-induced inhibition of cell cycle progression markedly preceded recovery from inhibition of [3H]TdR incorporation suggesting that nucleotide pools are markedly perturbed in HN2-treated cells. The difference in sensitivity of these two cell lines cannot be adequately explained by differences in amounts of initial DNA damage, rates of repair, differential S-phase delay or rate of loss of DNA crosslinks.

An SV40-transformed Fanconi's anaemia (FA) cell line, GM6914, exhibits approximately 2.4-fold increased sensitivity to the cytotoxic effects of nitrogen mustard (NM) when compared with the normal line, MRC5-V1. Host cell reactivation of NM-treated plasmid has been investigated using transient expression vectors which contain the chloramphenicol acetyltransferase (CAT) gene. In both cell types there is a similar, dose-dependent reduction in CAT expression which correlates with an increase in NM-induced DNA-interstrand crosslinking. The data are consistent with two possible mechanisms for inactivation of the plasmid. Either a single crosslink anywhere within the plasmid is sufficient to prevent transcription of the cat gene. Alternatively, inactivation may result from some other more prevalent NM-induced lesions within the cat coding sequence.

We have previously reported that chloroethyl nitrosourea and nitrogen mustard bone marrow toxicity can be selectively reduced by placement of the cytotoxic group on specific positions of a glucose molecule. We have now synthesized and evaluated a new drug in which the mustard cytotoxic group is attached to the carbon-6 position of galactose (C6-GLM). C6-GLM, administered i.p. as a single 10% lethal dose of 15.5 mg/kg, produced a 121% increase in life span (ILS) in mice bearing the ascitic P388 leukemia, compared to a 60% ILS with a 10% lethal dose of nitrogen mustard (P less than 0.01). A single p.o. dose of C6-GLM, 16 mg/kg, produced an ILS of 58%. Against i.p.-implanted B-16 melanoma, i.p. C6-GLM produced a 56% ILS compared to 30% with an equitoxic dose of nitrogen mustard (P less than 0.01). The activity of the two drugs for Ehrlich ascites was comparable, with 60% survivors with the galactose mustard. A single 10% lethal dose of C6-GLM reduced the white blood cells to 74% of control; circulating granulocytes remained at 91% of initial values. With nitrogen mustard, the nadir white blood cell count was 57% of control with an absolute granulocyte count of 70% of initial values (P less than 0.01). The toxicity of melphalan was considerably greater, with a lower and more protracted while blood cell nadir and an absolute neutrophil count nadir of 49% of control. These findings paralleled the relative decrements in bone marrow DNA synthesis produced by the three drugs. Measurement of human bone marrow granulocyte-macrophage colony-forming units, following in vitro exposure to graded concentrations of the three mustards, confirmed the bone marrow sparing properties of C6-GLM. At the highest concentration, 1 X 10(-2) mM, the latter drug produced only a 33% reduction in colonies compared to a 75% reduction with nitrogen mustard and a virtual elimination of activity of colony-forming units with melphalan. The demonstration of antitumor activity, at least equivalent to nitrogen mustard, without the necessity of significant bone marrow toxicity supports the development of C6-GLM for clinical trials in humans.

In the Walker 256 rat mammary carcinoma cell line, WR, resistance to nitrogen mustards (NM) is accompanied by collateral sensitivity to chloroethylnitrosoureas (CENUs). DNA-interstrand cross-links, DNA-protein cross-links, and sister chromatid exchange (SCE) induction were assayed in WR and the parent cell line (WS) after treatment with nitrogen mustard (HN2), phosphoramide mustard (PM), chlorozotocin (CLZ) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Treatment of cells with HN2 caused extensive levels of cross-links, approximately 50% of which were DNA-interstrand, equal in both WR and WS, whereas PM caused no detectable cross-links in either cell line. CLZ induced low levels of DNA-interstrand cross-links, similar in WR and WS, but no DNA-interstrand cross-links could be detected in either cell line after treatment with CCNU. Both CLZ and CCNU induced low levels of DNA-protein cross-links in both cell lines, though higher in WR than WS. There was no difference in the rate of removal of HN2-induced DNA-interstrand or DNA-protein cross-links or total CLZ-induced cross-links by the two cell lines, suggesting that differential repair was not relevant to the expression of resistance. Both HN2 and PM caused more SCEs in WS than in WR, whereas CLZ and CCNU induced more SCEs in WR. Thus, NM-induced SCEs were related to cell killing but not cross-linking, whilst CENU-induced SCEs were related to cell killing and DNA-protein but not DNA-interstrand cross-links. Furthermore, the collateral sensitivity of WR cells to CENUs was not due to the differential induction of DNA-interstrand cross-links or repair of total cross-links, or repair of total cross-links, although higher levels of DNA-protein cross-links occurred in WR, and these may be either a cause or a consequence of increased susceptibility of these cells to CENUs. Presumably NMs and CENUs have several distinct and separate macromolecular targets which result in differential cell killing. It is concluded that a range of lesions occurred after treatment of WR and WS cells with either NMs or CENUs and that, in these cell lines, there is no simple correlation between drug-induced cross-linking, SCE induction and cytotoxicity.

Levels of glutathione (GSH) and two enzymes involved in GSH metabolism, glutathione reductase (GR) and glutathione-S-transferase(s) (GST), were measured in four SV40-transformed human fibroblast cell lines. MRC5-V1 and GM0637, derived from normal individuals, had mean GSH levels of 4.2 and 6.5 nmoles/10(6) cells, respectively. TAT2SF and AT5BIVA, both from ataxia-telangiectasia (A-T) patients, respectively had 6.5 and 4.2 nmol/10(6) cells, indicating that basal GSH levels were similar in A-T and normal cells. There was some variation in GST activity among the four cell lines but deficiency in this enzyme cannot be associated with radiosensitivity in A-T. When GR activity was measured, A-T cells had approximately 82 per cent of the mean normal activity. Though statistically significant, (P = 0.05), this small deficiency could be due to chance and is unlikely to be responsible for the radiosensitive phenotype of A-T.

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

SK&F 102912 (mu-[1,2-bis(diphenylphosphino)ethane]bis[(1-thio-beta-D- glucopyranosato-S)gold(I)], [(Autg)2(dppe)]) has shown reproducible and significant activity in transplantable murine tumor models and represents a structurally unique class of antineoplastic agents. A number of in vitro studies were performed to elucidate the cellular pharmacology of this gold-containing complex. [(Autg)2(dppe)] is a potent cytotoxic agent in vitro as demonstrated by its ability to inhibit the clonogenic capacity of a variety of tumor cell lines following a brief exposure to the drug. Cell-cycle analysis using HL-60 cells showed that low concentrations (2 microM) of [(Autg)2(dppe)] induced an S-phase block and higher concentrations induced a secondary block at the G1/S boundary. [(Autg)2(dppe)] had several effects on DNA metabolism and structure including preferential inhibition in cells of DNA synthesis (relative to RNA and protein synthesis) and the production of DNA single- and double-strand breaks as measured by alkaline elution. The cytoxic mechanism of this gold complex appears to be distinct from that of the monophosphine-gold complex auranofin.

Previous studies have suggested that 1-(4-amino-2-methylpyrimidine-5-yl)-methyl-3-(2-chloroethyl) -3-nitrosoureahydrochloride (ACNU) and 1,(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) bind specifically to the nucleosomal DNA of murine bone marrow and L1210 leukaemia cells whereas the glucose nitrosoureas, 2-(3-(2-chloroethyl)-3-nitrosoureido)-2-deoxy-D-glucopyranose, (chlorozotocin, CLZ) and 1-(2-chloroethyl)-3-(-D-glucopyranosyl)-1-nitrosourea (GANU), bind preferentially to the linker DNA of bone marrow but not tumour cell chromatin. In order to provide an explanation for this differential, the DNA repeat and linker lengths in murine bone marrow and L1210 leukaemia cells were measured using electrophoresis of micrococcal nuclease-digested DNA. The linker length of bone marrow chromatin was approximately 22% longer than that in L1210 leukaemia cells from mouse ascites. The linker length of L1210 cells maintained in suspension culture was 27% less than in those from ascites fluid. The tissue-specific toxicity of sugar nitrosoureas and the differential binding of these drugs to chromatin does not appear to correlate quantitatively with differences in DNA linker length.

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.

A novel nitrosourea, 1-(2-chloroethyl)-3-[2-(dimethylaminosulfonyl)ethyl]-1-nitrosourea (TCNU) has been investigated with respect to cytotoxic mechanisms in rat and human cell lines which either possess (Mer+) or lack (Mer-) 0(6)-alkylguanine transferase activity. TCNU produced significantly greater cytotoxicity in the Mer- cells (Walker 256 rat breast carcinoma resistant to nitrogen mustards; human lung carcinoma A427) than in the Mer+ cells (Walker 256 wild-type; human lung carcinoma A594). This correlated with results generated by alkaline elution studies which showed that TCNU caused DNA interstrand crosslinks in A427 but not in A549 cells. Inhibition of glutathione reductase activity by TCNU demonstrated that in carbamoylating activity the drug was intermediate between chlorozotocin and 1,(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in both A427 and A549 cells. These data suggest that the presence of taurine in the drug structure does little to alter the cytotoxicity or the alkylating or carbamoylating properties of TCNU, and that any clinical advantages with TCNU will be the consequence of other factors.