Short-term exposure of rodents to diesel exhausts: usefulness for studies of genotoxic and immunotoxic effects.

An exposure facility was tested with regard to the information obtainable from short-term animal experiments for the assessment of health hazards from automotive engine exhausts. Indicators of immunotoxicity and genotoxicity were studied in guinea pigs and mice, respectively, exposed for 2 weeks, 8 h/day, to ten times diluted exhausts from a one-cylinder research diesel engine running at constant load. Regulated and non-regulated pollutants were determined. Besides increased number of lavageable cells in the airways, exposed guinea pigs exhibited, after immunization and challenge to ovalbumin, reduced leukotrienes B4 and C4 in lavage fluid and reduced anti-ovalbumin IgG in serum. Absence of increased CYP1A activity indicated that the exposure was below the threshold for induction of these enzymes. Instead a certain reduction of this activity indicated interaction with active enzyme sites. In vivo doses of some reactive metabolites of low molecular mass were measured by adducts to hemoglobin. Doses from aliphatic epoxides were low, in accordance with low hydrocarbon levels in the exhaust. The levels of hemoglobin adducts from aldehydes showed no clearcut influences of exposure. Genetic effects determined by DNA fingerprint analysis were indicated. It is concluded that repeated dose inhalation exposure of small numbers of animals is a useful mode of exposure for studying parameters that may elucidate toxic effects of air pollutants emitted from automotive engines, with a possibility to evaluate engine and fuel with regard to health hazards.

Male-male competition and female choice in brown trout.

In some salmonid species, the females have been assumed to choose their mates on the size of the male's adipose fin. This hypothesis was tested in a stream water aquarium, in which 19 brown trout, Salmo trutta, females were allowed to choose between two males of the same body size but with different adipose fin sizes. The two males were separated from each other in cages. After the female had started to prepare her nest close to one of them, the males were released and allowed to fight each other for the opportunity to spawn. Out of 19 females, 14 prepared a nest closest to the male with the larger adipose fin. However, only six of the 14 females spawned with this male. Males that spawned were more dominant (i.e. were more likely to win fights). When the female spawned with the male she chose, he was less aggressive towards her than when she spawned with the other male. There were no significant differences in the plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) between the chosen males and those not chosen. However, the dominant males had significantly higher plasma levels of T and 11-KT both before and after the experiment. The results support the view that female brown trout exhibit mate choice, but their choice is overruled by male-male competition. Copyright 1999 The Association for the Study of Animal Behaviour.

Polychlorinated biphenyls induce meiotic length mutations at the human minisatellite MS32 in yeast.

Polychlorinated biphenyls (PCBs) are lipophilic compounds, several of which are toxic and carcinogenic. Complex mixtures of PCBs (e.g., Aroclor) have been widely used in the industry. The persistence of PCBs, in combination with poor waste management, has led to a large-scale distribution of PCBs in the biosphere. The toxic and carcinogenic effects of PCBs are poorly understood, but are suggested to be associated with Ah receptor binding and induction of the Ah-gene battery. We have previously shown that a higher-chlorinated PCB mixture, Aroclor 1254, significantly increased the germline mutation rate at the mouse minisatellite PC-1. We have recently developed an in vitro model system to study and characterize spontaneous and induced meiotic mutations in human minisatellites integrated in yeast. Here, for the first time, we have used this model system to show that chemicals, in this case Aroclor 1254, can induce meiotic length mutations at the human minisatellite MS32 in a yeast strain harboring 38- and 42-repeat-unit alleles. The results also show that the size distribution of mutant MS32 alleles differs between PCB and the control, with a larger proportion of mutant allele sizes below 29 repeat units in the PCB series. These alleles were not structurally different from the alleles of the same size in the control. We conclude that PCBs induce minisatellite mutations in meiosis and have recombinogenic properties, and that the mutations are induced in an Ah receptor-independent manner. The induction of minisatellite mutations in meiosis as an indication of genomic damage must be taken into account in the risk assessment of PCBs and other environmental contaminants.

Induction of germline-length mutations at the minisatellites PC-1 and PC-2 in male mice exposed to polychlorinated biphenyls and diesel exhaust emissions.

PC-1 and PC-2 are hypervariable mouse minisatellites. The rates of spontaneous germline-length mutation have been shown to vary between different mouse strains. PC-1 is composed of GGCAG repeat units and PC-2 of GGCAGGA. Minisatellites frequently mutate by gaining or losing repeat units. Such length mutations in mini- and microsatellites have been associated with human disease and may therefore be an important endpoint in genetic toxicity testing. Carcinogenic activity of many chemicals is associated with their ability to induce heritable mutations. Since minisatellites are highly prone to mutate to new lengths, which can be assayed by Southern analysis, we used this method to detect heritable genetic effects in mice. Male mice exposed to diesel exhausts and/or polychlorinted biphenyls (PCB) were investigated for effects on the germline mutation frequenallele lengths in parents and offspring. For PC-1 significantly higher mutation frequencies were found in males treated with diesel exhausts + PCB (6 of 35 alleles) and with PCB alone (6 of 51 alleles) as compared to the males in the control group (0 of 43 alleles). The mutation frequency in the diesel exhaust group was not significantly increased (2 of 43 alleles). For PC-2 the only mutation found occurred in the PCB group (1 of 51 alleles). This in vivo study demonstrates--for the first time--chemically induced minisatellite mutations in the germline.

Chemically induced changes in the spectrum of amplifications of the human minisatellite MS1 integrated in chromosome III of a haploid yeast strain.

To study chemically induced DNA amplifications we used the haploid Saccharomyces cerevisiae strain TR(MS1)-1 carrying an integrated chromosomal copy of the human minisatellite. MS1. Chemicals with different mechanisms of action were tested in this strain: methyl methanesulphonate, ethylene oxide (EO), propylene oxide (PO), camptothecin, 2,3,7,8-tetrachlorodibenso-p-dioxin (TCDD) and reserpine. No increase in frequency of new MS1 length alleles was seen with any of the tested chemicals relative to the spontaneous frequency of approximately 30%. EO and TCDD induced changes in the amplification spectrum, i.e., the frequency distribution of MS1 length alleles longer than the original 1.42 kb allele. PO and camptothecin increased the frequency of plasmid "pop-out" events. It seems likely that several mechanisms e.g. unequal exchanges, replication slippage and loop formation leading to deletion of a ring of tandem repeats, are involved in the generation of new MS1 length alleles. A loop-forming deletion mechanism is supported by the tendency to multimodality shown in the deamplification (loss of repeat units) spectra, i.e. the frequency distribution of new MS1 length alleles shorter than the original allele. EO and TCDD induced "longer" MS1 length alleles as compared to the control. The frequent generation of new MS1 length alleles in this haploid yeast strain further demonstrates the instability of such sequences and their possible relevance to genetic toxicology and the mechanisms of induction of cancer as well as other diseases. This study is a first step towards the development of an assay for DNA amplification without the use of a selective agent.

Amplification and loss of repeat units of the human minisatellite MS1 integrated in chromosome III of a haploid yeast strain.

Minisatellites comprise arrays of tandemly repeated short DNA sequences which show extensive variation in repeat unit number. The mechanisms that underlie this length variation are not understood. In order to study processes influencing length changes of minisatellites, we integrated the human minisatellite MS1 into a haploid strain of the yeast Saccharomyces cerevisiae. Frequent spontaneous generation of MS1 alleles with new lengths were observed in this yeast strain. Hence it is concluded that recombination between members of a pair of homologous chromosomes is not a prerequisite for the generation of length changes in MS1 in yeast.

Differences in c-myc and pvt-1 amplification in SEWA sarcoma sublines selected for adherent or non-adherent growth.

Conversion of solid sarcomas and carcinomas into ascites tumors depends on the in vivo selection of phenotypically altered tumor cell variants that can grow in the dissociated form. Once selected, they retain this property even after prolonged s.c. growth as solid tumors. From an s.c.-passaged subline of an ascites-converted murine sarcoma (SEWA-AS12), we were able to separate cells adapted to the ascites form of growth from cells that can only grow in the solid form on the basis of their differential adherence to plastic. Both c-myc and pvt-1 were amplified approximately 63- to 77-fold in the nonadherent subline (SEWA-AS12-NA), but only 5- to 8-fold in the adherent subline (SEWA-AS12-ADH). This suggests that c-myc and/or pvt-1 amplification may provide a selective advantage to cells that can grow in the dissociated form.

High prevalence and high titers of LAV/HTLV-III antibodies in healthy hemophiliacs in the midwestern United States.

Twenty-eight patients from the Nebraska Regional Hemophilia Center were studied for the prevalence and titers of antibodies to lymphadenopathy-associated virus/human T cell lymphotropic virus type III (LAV/HTLV-III) and for clinical symptoms of possible progression to the acquired immune deficiency syndrome (AIDS). Ten of 18 (56 percent) patients with hemophilia A who were frequently treated with commercial factor VIII concentrate were seropositive for LAV/HTLV-III antibodies as determined by immunofluorescent study and Western blot testing. Of the four factor VIII-deficient patients who were seronegative, one had received only heat-treated factor VIII concentrates, two had received only cryoprecipitate, and one had received no transfusions since 1983. None of the patients treated only with factor IX concentrate, volunteer donor plasma, or cryoprecipitate had LAV/HTLV-III antibodies. In nine of 10 seropositive hemophiliacs, titers of serum antibodies to LAV/HTLV-III ranged from 1:1,280 to 1:10,240, indicating a strong immune response against LAV/HTLV-III antigens and/or persistent infection with the virus. Serum from seropositive hemophiliacs interacted on Western blot testing with all the major LAV/HTLV-III polypeptides, including envelope proteins gp 42 and gp 120. Despite the possible exposure to LAV/HTLV-III during the past four years, none of the patients in this group had symptoms suggestive of progression towards AIDS. Whether or not immunity to the AIDS retrovirus developed in this group of patients remains to be determined.

Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, has the ability to transform human B lymphocytes. No other cell type has been experimentally transformed by EBV, either by intact virions or naked viral DNA and subgenomic fragments. Two immortalized human T-lymphoblastoid cell lines have now been established by transfecting cord blood lymphocytes with purified B95-8 viral DNA enclosed in fusogenic Sendai virus envelopes (RSVE) and then exposing the cells to EBV from a P3HR-1 cell subclone. One of these lines, which has been fully characterized, is termed HBD-1. This line is positive for EBV DNA and expresses surface OKT11, OKT4, and Tac receptors, but not M-1, mu immunoglobulin chains, EBV receptors, or B-1 surface markers. The cells contain fully rearranged T-cell receptor genes and germline immunoglobulin genes. The karyotype of the cells is normal, they do not require interleukin-2 for growth, and do not contain human T-lymphotropic virus type I. However, the HBD-1 cells contain incomplete EBV genomes and express several EBV-determined antigens, including the early antigen type D, membrane antigens, but not EBV-determined nuclear antigen (EBNA). This association of the EBV genome with permanently growing hematopoietic cells of non B-cell lineage should prove useful in studies on the mechanism of EBV-mediated cell transformation.

Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications.

Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus-producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV-III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes.

[Endogenous variations of productivity in Scenedesmus acutus and their relation to the nucleic acid metabolism]. / Endogene Produktivitätsschwankungen bei Scenedesmus acutus und ihre Beziehung zum Nucleinsäure-Stoffwechsel.

1. In Scenedesmus acutus Tomaselli, endogenous variations in cell progeny production and chlorophyll formation have been found which are very similar to those previously described in Chlorella by Hesse (Z. Pflanzenphysiol. 67, 58-77, 1972). When the dark phase of the light-dark-cycle is prolonged to a certain extent, cell productivity drops to a minimal value during the next normal light-dark-cycle. If the duration of the supplementary dark treatment comes near to 24 h, cell productivity is almost normal during the next cycle. 2. Nucleic acid labeling with radioactive precursors is very similar in Scenedesmus acutus and Chlorella pyrenoidosa. Short time labeling with uridine results in labeled chloroplastic RNA and DNA, the cytoplasmic RNA being almost unlabeled. With guanosine, both chloroplastic and cytoplasmic RNA as well as DNA are labelled. In nucleic acid separation on acrylamide gels special caution must be taken, since endocellular RNases are particularely active in some cell stages of Scenedesmus. Optimal results are obtained with ripe mother cells; during nucleic acid purification, cell homogenates have to be frozen together with the phenol-cresol mixture. 3. Large differences in guanosine incorporation are found after treatment of the cells with supplementary dark time. After the normal 10:12 h light-dark-cycle, and also after 24 h of supplementarry dark time, much more radioactive guanosine is incorporated into chloroplastic RNA than into cytoplasmic RNA. After 12 h of supplementary dark time, however, cytoplasmic RNA is more extensively labeled than chloroplastic RNA. 4. When the specific radioactivity of guanosine is diluted to one half, the incorporation into the rRNA of cytoplasm and chloroplast is strongly reduced. This is due to the filling up of the guanosine pool in the two compartments. In contrast, DNA labeling is hardly influenced by reduced specific radioactivity of the precursor. This may be interpreted as meaning that the radioactive labeling reflects the rate of DNA synthesis rather than the size of the guanosine pool in the nucleus. Differences found in the labeling of DNA after 12 and 24 h of supplementary dark time can than be interpreted as variations in DNA synthesis rate.