The genetics of a small autosomal region of Drosophila melanogaster, including the structural gene for alcohol dehydrogenase. V. Characterization of X-ray-induced Adh null mutations.

Of 31 X-ray-induced and 2 spontaneous Adh null mutations selected for resistance to pentenol (Aaron 1979), 21 are deletions, including Adh and one or more neighboring loci. By contrast, none of 13 EMS-induced Adhn mutations are deletions. On average, the size of these X-ray-induced deletions is shorter than that of 12 formaldehyde-induced Adhn deletions (O'Donnell, Mandell, Krauss and Sofer 1977). Both the X-ray- and formaldehyde-induced deletions show a nonrandom distribution of break points in region 34D to 35D of chromosome arm 2L. Some of the deletions display particular genetic properties associated with one of their end points.

End points for biomonitoring: assay sensitivity/selectivity.

Estimation of population exposure and biological impact of potential hazards are central reasons for performing biomonitoring. The sensitivity of the biomonitoring methods and the linkage of the measured phenomenon to human disease are also important, but often overlooked, considerations. We are conducting experiments to evaluate the sensitivity of hprt mutation measurement in the nonhuman primate, the cynomolgus monkey. Our findings demonstrate in the monkey that hypoxanthine guanine phosphoribosyltransferase (hprt) mutations produced in vivo can be detected using technique originally worked out using human cells; cynomolgus monkeys were chosen to avoid many of the complications encountered in studying humans. Sequencing of mutants from the monkey using reverse transcriptase polymerase chain reaction methods has led us to conclude that there is similarity of the spectra observed between the spontaneous mutations detected in the two species. However, more recent data suggest that due to low sensitivity, the method is probably not appropriate for routine biomonitoring of randomly selected populations. For example, the inability of the hprt mutation assay to detect some very potent mutagens in the monkey and the effects of the time-dependent pattern of mutant occurrence serve to urge caution in interpretation of elevation or lack of elevation in mutant frequency. Mechanisms for splitting and archiving samples of human tissues/blood from populations at risk may prove valuable as methods improve.

Risk and benefit evaluation in development of pharmaceutical products.

Pharmaceutical products are intended to cure disease, reduce pain and suffering, prolong life, and correct metabolic deficits in patients. However, the potential patient population is intrinsically genetically heterogenous, and this factor complicates the evaluation of data on all aspects of safety evaluation of new drugs. Often the genetic heterogeneity is related to drug metabolizing capacity, but recent evidence suggests that heterogeneity in repair capacity as well as structural integrity of the chromatin (fragile X) have been shown to be relevant. Because drugs are biologically active and may have more than one type of effect, the evaluation of a large number of parameters is necessary in arriving at a rational estimate of potential risk. In this paper, several specific examples of risk assessments and some generic genotoxicity questions that are recurrent, including the question of the relevance of in vitro chromosomal aberration induction at high dose/sampling time, are raised. Other examples of the kinds of concerns from the safety evaluation of U-48753E, U-54461, and U-68,553B are discussed. The drug U-48753E was discovered to be slightly mutagenic in the AS52 assay, and significant efforts were expended in evaluation of the metabolism-based generation of a reactive intermediate. The drug U-54,461 was shown to be capable of breaking chromosomes in vitro but extensive in vivo data as well as a variety of other studies served to reduce the level of concern substantially.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo mutagenesis in the cynomolgus monkey: time course of HPRT mutant frequency at long time points following ethylnitrosourea exposure.

We have monitored mutant frequency at the HPRT locus in peripheral blood lymphocytes of cynomolgus monkeys using a clonal assay in which mutants are selected by resistance to 6-thioguanine. Among untreated animals, the mean spontaneous mutant frequency was 2.9 +/- 2.9 x 10(-6) (standard deviation, based on 131 determinations in 33 animals), in good agreement with HPRT mutant frequencies in other species. In four animals treated with a single intraperitoneal dose of 77 mg/kg ethylnitrosourea, mutant frequency increased with time, peaking 70 to 100 days after treatment. Mutant frequency in two of the four animals was monitored at intervals for 6 years, and a second identical treatment was given about 830 days after the first. Mutant frequency again peaked in these two animals 70 days after the second dose and decreased following peak values, declining to a plateau that was higher than the predose mutant frequency in both animals. This pattern was repeated following the second ethylnitrosourea treatment. Fractionating the dose of ethylnitrosourea into five equal daily injections had no effect on mutant frequency in two animals when compared to a single dose.

DNA sequence analysis of spontaneous and N-ethyl-N-nitrosourea-induced hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.

The study of hprt mutations in cynomolgus monkey T-lymphocytes is part of our effort to understand the mechanisms of mutagenesis in vivo. This primate model allows us to study mutations and their kinetics at the molecular level under well-controlled conditions using recently developed techniques for selection of mutant T-cells and polymerase chain reaction (PCR) amplification of hprt cDNA, which is directly sequenced. This is the first report of the sequence of the coding region of the cynomolgus monkey hprt gene and PCR/DNA sequence analysis of seven spontaneous mutant T-cell clones, as well as 23 mutant clones isolated 63 and 601 days after treatment with ethylnitrosourea (ENU, 77 mg/kg, intraperitoneal). cDNA was reverse transcribed from hprt mRNA directly from a lysate of about 2-4 x 10(3) cells, and a 700 bp fragment including the coding region was amplified by PCR and sequenced. Of the seven spontaneous mutants, only one point mutation (GC----AT transition) was detected, and the other six failed to amplify by PCR, possibly due to functional deletions. Of the 14 mutant clones isolated 63 days after ENU treatment, nine base substitutions were detected in ten clones: four transitions (three AT----GC and one GC----AT) and five transversions (four AT----TA and one AT----CG). Of the nine mutants isolated 601 days after ENU treatment, six single base substitutions were detected in six clones (five AT----TA and one AT----CG transversions), and one mutant had a large deletion or insertion. No changes were detected in three clones (one Day 63 and two Day 601 clones). In summary, only one of 15 single base substitutions isolated after ENU treatment was a GC----AT transition mutation and the rest were transitions and transversions at AT sites.

Chromosomal breakage following treatment of CHO-K1 cells in vitro with U-68,553B is due to induction of undercondensation of heterochromatin.

Preferential breakage of chromosomes at specific sites (so-called "fragile sites") has been observed to occur spontaneously, and has been induced by some metal salts and chemicals. Furthermore, a heterochromatic region of the long arm of the Chinese hamster ovary (CHO) X-chromosome is known to be susceptible to a disproportionately high frequency of spontaneous breakage; unless there is physical displacement of chromatin the resulting achromatic lesions are not scored as structural aberrations. We have encountered such anomalous breakage associated with C-band positive regions of the chromosomes of a CHO-K1 cell line following exposure of the cells to toxic doses of U-68,553B and in this report present evidence that the apparent breaks are due to undercondensed heterochromatin (UH) and evidence that the phenomenon appears to occur at higher frequency in a particular cell line of Chinese hamster. This finding has important implications on the assessment of potential risk due to exposure to the drug. Such apparent breaks at sites of UH in chromosome 1 was not observed in an alternate CHO cell line (CHO-WBL) which supports the notion that the UH associated achromatic lesions in the CHO-K1 line may be a cell line specific phenomenon. Furthermore, careful electron microscopy of the chromosomes revealed chromatin fibers connecting the apparently broken chromosomes. The UH was not observed in the presence of added metabolic activation (S9), and thus the significance of the phenomenon in risk assessment is further reduced. The data presented here provide evidence that sites of UH occur preferentially at locations of C-band positive constitutive heterochromatin in CHO cells; we believe that this is the first report of induced fragile sites in rodent cells in vitro documented in this way. In addition, evidence is presented that U-68,553B lacks the ability to induce breakage in vivo in rodents and lacks the ability to induce chromosome breakage in human peripheral lymphocytes in vitro. Therefore, it is concluded that the positive results with CHO-K1 cells treated with U-68,553B are unlikely to be predictive of a genotoxic hazard. This is a specific example of the importance of careful followup to an in vitro result in risk assessment.

Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacI transgene in the Big Blue rat-2 embryo cell line.

Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5). Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

Enumeration of 6-thioguanine-resistant T-lymphocytes in the peripheral blood of nonhuman primates (cynomolgus monkeys).

We have investigated the use of cynomolgus monkeys (Macaca fascicularis) as a model of somatic cell mutagenesis in non-human primates. Using techniques described by Albertini (Mutation Research 150:411-422, 1985) for similar studies in humans, the frequency of TG-resistant T-lymphocytes in the peripheral blood was determined in animals that were either untreated or treated with ethylnitrosourea. The frequency of TG-resistant cells in untreated males was (mean +/- SD) 6.0 +/- 5.9 per 10(6) cells and for untreated females was 2.9 +/- 2.7 per 10(6) cells. The spontaneous frequency of TG-resistant cells for all animals was 4.2 +/- 4.44 per 10(6) cells. Maximum frequency of TG-resistant cells for two animals treated with a single I.P. dose of ENU was 45.1 and 77.9 per 10(6) cells. Substantial increases in frequencies of TG-resistant cells were not seen until at least 63 days after treatment. The TG-resistant phenotype of clones isolated in the assay was stable after growth for 2 weeks in the absence of selective agent. Many of the TG-resistant clones selected were frozen for future molecular analysis.

Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay.

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.

Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073-9078] to the previously established Big Blue assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 x 10(-5) for cII/cI vs. 15.1 x 10(-5) for lacI; spleen = 64.8 x 10(-5) for cII/cI vs. 36.1 x 10(-5) for lacI; lung = 17.1 x 10(-5) for cII/cI vs. 15.8 x 10(-5) for lacI). Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs. 2.7 for cII/cI; spleen = 13.1-fold for lacI vs. 8.4 for cII/cI; and lung = 5.6-fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated.

DNA sequence analysis of spontaneous hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T-lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wild-caught cynomolgus monkey peripheral T-lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6-thioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780-base-pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monkeys. Of these 40 clones, 23 (57%) had base-pair substitutions, 11 (28%) had small (< 20 bp) deletions and/or insertions, and 6 (15%) had large (> 20 bp) deletions and/or insertions. Of the 23 base substitutions, 13 were transitions (11 G:C-->A:T, 1 A:T-->G:C, and 1 tandem TT-->CC) and 10 were transversions (3 G:C-->T:A, 3 G:C-->C:G, 2 A:T-->T:A, 2 A:T-->C:G). Bases 209 and 617 were apparent substitution hotspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic origin. One of these lost 272 bp from exons 2-3 and contained a 93-bp insertion from the middle of intron 3. Two clones with small deletions and 5 clones with large deletions or insertions (7/40 or 17.5%) could be splice mutants.

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed.

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice.

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

Micronuclei in mice treated with monocrotaline with and without phenobarbital pretreatment.

Monocrotaline is a very potent toxin, producing significant effects of pneumotoxicity, hepatotoxicity, and teratogenicity, as well as carcinogenicity. In addition, the compound has been clearly shown to be mutagenic after metabolic activation. The goal of the experiments reported here was to confirm the reported clastogenesis induced by this agent in vivo and to evaluate the impact of modulation of metabolic activity by phenobarbital, a potent P-450 inducer (both Phase I and Phase II enzymes). The method used in addressing this problem relied on a new technique for monitoring clastogenesis in vivo, i.e., the acridine orange micronucleus assay method originally exploited by Hayashi et al. [1990]. The result of our experiments confirmed monocrotaline to be an effective clastogen in vivo, using the acridine orange method of assessment. The peak in induction of micronuclei occurred on the second day following intraperitoneal administration of the drug. Administration of phenobarbital prior to monocrotaline did appear to modulate the micronucleus induction. At 30 mg/kg bw monocrotaline, the pretreatment with phenobarbital appears to increase the intensity of monocrotaline clastogenesis, while the effect at higher doses (60 and 125 mg/kg bw) is a reduction in potency, presumably reflecting increased importance of Phase II metabolism for monocrotaline at these doses. Thus the study reported here confirms the potent in vivo clastogenesis of monocrotaline, and provides evidence for a dose-related shift in mechanism for the phenomenon.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys.

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.), chloroethylmethane sulfonate (CI-EMS, 35 or 50 mg/kg i.p.), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 microg/kg i.v.) and CC-1065 (6 microg/kg i.v.) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg i.v.) were equivocal. Adozelesin, CC-1065, and CI-EMS treatments increased the percentage of T-lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys.

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.

Antagonism of kainic acid lesions in the mouse hippocampus by U-54494A and U-50488H.

A morphometric study of kainic acid- (KA) induced lesions was designed for the study of the interaction of the diamines U-5449A and U-50488H with excitatory amino acids, and the dose-response relationship thereof. IC50S determined for binding at the kappa receptor and other opioid receptors demonstrated the lack of kappa activity of U-54494A, a structurally related analog of U-50488H. Both opiate kappa receptor related anticonvulsant diamines were tested for their ability to protect the mouse hippocampus from the cytopathological changes induced by KA in neurons and glia. The damage observed with i.c.v. KA in mouse was restricted to neurons of the CA3 pyramidal region and glia of the hippocampus. It involved massive cell loss and shrunken neurons with dark cytoplasm and nuclei. Groups treated with combinations of KA and U-54494A or U-50488H showed scarce damage, but patches of necrotic changes were still observed. Control animals treated with saline (i.c.v.) and U-54494A (s.c.) or U-50488H (s.c.) did not suffer any noticeable alterations of the polymorphic layers of the hippocampal formation. Image analysis of the CA3 area of the hippocampus was used to quantitate the vacuolization induced by KA lesions in the control and treated groups. By this method, both U-54494A and U-50488H were shown to protect this area in a dose-related fashion as evidenced by reduced vacuolization. The anticonvulsant properties of these compounds may result in the antagonism of the excitotoxic lesions. More specifically, the ability of these diamines to block depolarization-induced influxes of Ca++ may protect the CA3 cells from the cytotoxic effects of persistent depolarization.

X-ray-induced mutations affecting the level of the enzyme alcohol dehydrogenase in Drosophila melanogaster: frequency and genetic analysis of null-enzyme mutants.

The frequency of X-ray-induced (null-enzyme) mutations at the alcohol dehydrogenase locus in Drosophila melanogaster was measured. The rate of recovery of chromosomes that fail to direct the synthesis of a functional Adh protein is 3 x 10(-8) per R for chromosomes that do not include large chromosome rearrangements. However, this analysis excludes a larger number of chromosomes that are "null-enzyme mutations" because thye are deleted for the region of the Adh locus. The dose of X-rays required to induce a frequency of non-deletion null-enzyme mutants equal to the spontaneous frequency is about 73 rad calculated from the data reported in this communication.

X-ray induced sex-chromosome loss, when ring-X chromosome males are irradiated and mated to females carrying mei-9, mei-41 or mei-218.

Radiation damage induced in the sperm nucleus is repaired after this nucleus has entered the oocyte. The yield of induced genetic damage is known to be dependent on maternal genotype and can also be modified by treatment of the females with metabolic inhibitors. The experiments reported here were designed to find out whether a more specific analysis of the interaction between male gamete and oocyte cytoplasm can be carried out using mutants that are known to affect repair processes. Males carrying ring-X chromosomes were exposed to X-ray doses up to 1000 R and mated to females homozygous for a repair-deficient mutant. The mutants used were mei-9a, mei-9L1, mei-41A10, and mei-41D5. In addition a yellow (y) X chromosome was used as a control and an attempt was made to obtain data using mei-21815, a mutant at a locus not thought to affect repair. With mei-9 mutants there is an enhancement of the spontaneous and induced rates of paternal sex-chromosome loss. The mei-41 mutants did not affect the rates of paternal sex-chromosome loss. Mei-218 females were difficult to work with because they gave very few progeny. From these data it can be argued that repair-deficient mutants will indeed be useful for an analysis of the fixation of radiation-induced genetic damage.