Presence of DNA of adeno-associated virus in subfertile couples, but no association with fertility factors.

BACKGROUND: Based on previous reports suggesting a role of adeno-associated virus (AAV) in miscarriage, the prevalence of AAV DNA in genital tracts of male and female partners of subfertile couples was determined to assess a potential association of AAV infection with clinically relevant parameters of male and female fertility. METHODS: A prospective study was performed in the outpatient infertility clinic of a university-based hospital. Semen samples and endocervical material obtained from 146 male and 134 female partners of asymptomatic subfertile couples were analyzed for the presence of AAV DNA (using nested PCR). Patients' medical histories and details of clinical examinations were recorded. Semen quality, including sperm functional capacity and the presence of antisperm antibodies (ASA) and seminal white blood cells (WBC), was assessed in aliquots of the same ejaculate. Detailed examinations of the cervical factor and other variables of female subfertility were performed. Both partners were screened for bacterial infection. RESULTS: The presence of AAV DNA in semen was not significantly related to semen quality, including sperm functional capacity or local ASA, nor was it coupled to the presence of AAV in the endocervical material of female partners. The presence of AAV DNA was not associated with the presence of other micro-organisms of the lower genital tract or with seminal WBC in men. AAV DNA in endocervical material was not related to a reduced quality of cervical mucus or to other female infertility factors. CONCLUSIONS: The presence of AAV DNA in semen samples or endocervical swabs showed no significant association with clinically relevant infertility factors. However, longitudinal studies may clarify previous suggestions of an influence of AAV infection on early pregnancy problems.

DNA amplification of adeno-associated virus as a response to cellular genotoxic stress.

We studied DNA amplification of helper virus-dependent parvoviruses [adeno-associated virus (AAV)] following genotoxic treatment of a number of mammalian cell lines from different species including primary, immortalized, and tumorigenic cells. All cell lines, either infected with AAV or transfected with parvoviral DNA, readily amplified AAV DNA in the absence of helper virus following treatment of cells with a wide variety of genotoxic agents like chemical carcinogens, UV, heat shock, and metabolic inhibitors of DNA replication or protein synthesis. In addition, we show that in the SV40-transformed Chinese hamster cell lines CO60 and CO631 carcinogen-induced AAV DNA amplification may result in a complete AAV replication cycle giving rise to infectious AAV progeny. Our results demonstrate that AAV DNA amplification induced by genotoxic agents is completely independent of the presence of viral helper functions. Because its induction is not restricted to a specific cell type or to a malignant phenotype, AAV DNA amplification may represent a marker for cellular genotoxic stress response.

Enhanced sensitivity of small cell lung cancer cell lines to cisplatin and etoposide after infection with adeno-associated virus type 2.

In previous studies we have reported the sensitisation of human tumour cells to gamma irradiation and chemotherapeutic drugs upon infection with the human non-pathogenic adeno-associated virus type 2 (AAV-2) in vitro and in vivo. Treatment of small cell lung cancer (SCLC) is consistently hampered by relapses due to the selection of chemotherapy-resistant cell clones. Hence, we were interested to test whether selection of chemotherapy-resistant SCLC cells might be reduced or even prevented if chemotherapy is applied in combination with AAV-2 infection. In vitro proliferation assays indicated that the number of proliferating cells, after combined treatment with cisplatin and etoposide, can be significantly reduced by concomitant AAV-2 infection, as compared with treated but non-infected controls. H446 SCLC cells, which show resistance to etoposide/cisplatin chemotherapy (compared with a cell line which was never chemotherapeutically treated before, like NCI-H209) were significantly more sensitive after AAV-2 infection, suggesting that the therapeutic efficacy of chemotherapy in SCLC can be enhanced even if the cells are already relatively resistant to chemotherapy. Similarly, in vivo growth of tumours induced by inoculation of SCLC cells into immunocompromised nude mice was reduced more efficiently in AAV-2-infected animals compared with tumours in mice treated with chemotherapeutic drugs alone. These data extend and further support our previous reports on AAV functions which might be useful in improving the efficacy of chemotherapeutic drugs used in human cancer treatment.

Improved efficacy of chemotherapy by parvovirus-mediated sensitisation of human tumour cells.

Increasing resistance of tumour cells towards the cytotoxic action of chemotherapeutic drugs is a major limitation in the treatment of cancer patients. The non-pathogenic human adeno-associated viruses (AAV) have been reported to sensitise HeLa cervical cancer cells to gamma irradiation in vivo and in vitro. To test whether these parvoviruses might render other human tumour cells more sensitive towards chemotherapeutic drugs, we analysed the effects of AAV type 2 (AAV-2) infection on established cancer cell lines and freshly explanted tumour biopsies treated with chemotherapeutic agents (e.g. cisplatin). AAV-2 infection significantly increased the cytotoxic activity of chemotherapeutic drugs compared with uninfected controls. AAV-2 infection without concomitant chemotherapeutic treatment had no significant effect on viability of the cells. In nude mice, combined application of AAV-2 infection and chemotherapeutic treatment significantly increased the therapeutic activity on tumours arising from subcutaneously injected tumour cells compared with tumours treated by chemotherapeutics only. These results indicate that AAV-2 infection sensitises human cancer cells towards the cytotoxic action of chemotherapeutic drugs.

Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells.

Adenoviruses, types 2 and 12 induce amplification of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/*PRF/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpes group or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatocellular carcinoma) remains to be determined.

Persistence of parvovirus H-1 DNA in human B- and T-lymphoma cells.

Persisting DNA of parvovirus H-1 could be demonstrated in cells of two human lymphoma cell lines, the Burkitt lymphoma cell line BL2 and the T-cell leukemia cell line Jurkat which survived infection with parvovirus H-1. Persistence of H-1 DNA rendered the cells resistant to a second H-1 infection. This resistance to H-1 superinfection persisted even after loss of H-1 DNA occurring after approximately 150-200 cell generations. Resistance to H-1 superinfection was accompanied by reduced uptake of infectious particles and by a block of H-1 DNA replication. This suggests that persistent H-1 infection leads to modifications of cellular functions involved in the permissivity for H-1.

Functional analysis of different LMP1 proteins isolated from Epstein-Barr virus-positive carriers.

The Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is implicated in the development of several human malignancies. Latent membrane protein 1 (LMP1), an EBV protein with known oncogenic properties, may be important in the pathogenesis of EBV-associated tumors, particularly nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD). Several reports suggested that sequence variations in the LMP1 gene may define a more aggressive, geographically restricted EBV-genotype. Most mutations in the LMP1 gene described are located within the C-terminus of the protein. However, the effect of these mutations on the biological function of the protein remains widely unknown. Therefore, this study aimed in investigating whether mutations detected in LMP1 genes isolated from different EBV-positive carriers have an effect on the biological function of the protein. For this purpose the LMP1 genes were amplified by nested PCR from DNA out of bone marrow and peripheral blood lymphocytes and sequenced. Three functional assays were performed in order to evaluate the biological activity of the different isolates: activation of the transcription factors NF-kappaB and AP-1 as well as the anchorage independent growth of LMP1 transfected ratl cells in soft agar. The results suggested that whereas differences in the activation of NF-kappaB through the various LMP1 isolates correlated tightly with their different expression levels, the outgrowth of transfected cells in soft agar did not and the transcription factor NF-kappaB therefore appeared not to be the major effector for the transformation of the rodent cell line ratl by LMP1. The various LMP1-isolates also differed in their capacity in activating the transcription factor AP-1. We found no correlation between the transforming ability of the LMPI isolates and activation of AP-1 suggesting that other so far uncharacterized domains also influence the transforming ability of the protein.

Adeno-associated virus DNA in human gestational trophoblastic disease.

Previous studies had shown a correlation between infection with the human adeno-associated virus (AAV) and spontaneous abortion in early pregnancy. Furthermore, AAV DNA had been detected in cells of the human trophoblast lines, Jeg-3, JAr, and BeWo, in cells of the human amnion line, FL, and in trophoblasts from amnion fluids. Infectious AAV virions could be isolated from amnion fluids. To further analyse AAV infection during pregnancy, we tested material from Gestational Trophoblastic Disease for the presence of AAV DNA. With 63 tissue samples from patients from Brazil, including 49 hydatiform moles and 14 choriocarcinomas, nested PCR was performed to detect the presence of AAV DNA. In addition, 15 samples from spontaneous abortions were analysed. AAV DNA was found in 43 samples (28/49 hydatiform moles, 4/14 choriocarcinomas, 11/15 miscarriage material). These findings confirm AAV infection of embryo-derived tissue in humans and further suggest a role of AAV in miscarriage and trophoblastic disease.

Biological activity of N-nitrosodiethanolamine and of potential metabolites which may arise after activation by alcohol dehydrogenase in Salmonella typhimurium, in mammalian cells, and in vivo.

The potent carcinogen N-nitrosodiethanolamine (NDELA) which is nonmutagenic in standard modifications of the S. typhimurium/mammalian microsome assay, can be activated effectively by alcohol dehydrogenase/NAD (ADH/NAD) to intermediates which are directly mutagenic in strains TA 98 and TA 100. The expected metabolites N-nitroso-2-hydroxymorpholine (NHMor), N-nitroso-(2-hydroxyethyl)-glycine (NHEG), N-nitrosoiminodiacetic acid (NIDA), and glycolaldehyde were assayed for their direct mutagenic activities in S. typhimurium TA 1535, TA 98, and TA 100. All compounds were clearly mutagenic in TA 100, but different specificities were observed for the other strains. NDELA and its putative mutagenic metabolites were also tested for induction of genotoxic activities by determination of DNA single strand breaks in primary rat hepatocytes. In these cells, NDELA and NHMor were clearly genotoxic, whereas NHEG and NIDA were inactive. In contrast, when assayed for the induction of selective DNA amplification NDELA and its metabolites were not found to induce SV40 DNA synthesis in SV40-transformed Chinese Hamster cells. The compounds were also assayed for induction of DNA single strand breaks in the liver after a single oral application to rats. NDELA and NHMor were about equally active in this in vivo test, whereas NHEG, NIDA and glycolaldehyde were inactive. Differences in biological activity in the cultivated cells, as compared to hepatocytes or to the in vivo situation may most probably be due to differences in metabolism and/or pharmacokinetics.

Genotoxic activities of benzamidine and its N-hydroxylated metabolite benzamidoxime in Salmonella typhimurium and mammalian cells.

The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.

Determination of DNA single strand breaks and selective DNA amplification by N-nitrodimethylamine and analogs, and estimation of the indicator cells' metabolic capacities.

N-nitrodimethylamine is metabolized oxidatively to N-nitrohydroxymethylmethylamine, which decomposes to yield formaldehyde and N-nitromethylamine. All four compounds and N-nitromethylamine were tested for their ability to induce DNA single strand breaks in hepatocytes and in SV 40-transformed Chinese hamster embryo cell lines. Only the two monoalkylnitramines were positive. They induced single strand breaks in hepatocytes, but were not effective in the other cells. Formaldehyde and N-nitrohydroxymethylmethylamine were toxic to the cells. None of the compounds tested was able to induce selective DNA amplification in the two transformed cell lines. Enzymes involved in drug metabolism were assayed in the hamster cell lines. The activity of UDP-glucuronosyltransferase and cytosolic epoxide hydrolase were not detectable. N-nitrodimethylamine demethylation was low. The content of reduced glutathione and the activities of glutathione transferase and membrane bound epoxide hydrolase were comparable to values obtained in the rat liver.

Discrimination between different types of human adeno-associated viruses in clinical samples by PCR.

Persistent infection of human tissues with the helper virus-dependent parvovirus, adeno-associated virus (AAV) was detected by polymerase chain reaction (PCR) using primer pairs detecting AAV types 2, 3 or 5. In order to develop PCR protocols which discriminate between the different serotypes of AAV, the DNA of AAV-5 was sequenced partially and compared with the published sequences of AAV-2 and -3. Type specific oligonucleotides and specific probes which allow the distinction between human AAV types by PCR are described.

Detection of adeno-associated virus in human semen: does viral infection play a role in the pathogenesis of male infertility?

OBJECTIVE: To evaluate the occurrence of adeno-associated virus (AAV) DNA and/or human papillomavirus (HPV) DNA in the semen of infertile men as a possible factor in the pathogenesis of male infertility. DESIGN: Descriptive pilot study. SETTING: University-based diagnostic and research laboratory. PATIENT(S): Semen specimens were collected from 30 men with diagnosed infertility and from 8 control subjects. INTERVENTION(S): Diagnostic spermiograms were made and the semen specimens were separated into seminal fluid, nonspermatozoal cells, and spermatozoa using a Ficoll gradient technique. MAIN OUTCOME MEASURE(S): The presence of AAV and HPV DNA in the different fractions of the ejaculates from the infertile men and the control subjects was detected by polymerase chain reaction. Semen quality was analyzed according to World Health Organization guidelines. RESULT(S): Adeno-associated virus DNA was detected in 30% (9/30) of the ejaculates from the infertile men. No AAV DNA was found in the ejaculates from the 8 control subjects. In 8 of 9 samples, AAV DNA could be found only in the spermatozoal fraction of the specimen. Seven of 9 semen specimens that contained viral DNA also demonstrated oligoasthenozoospermia. Both AAV and HPV DNA was found in the spermatozoal fraction of 3 of 30 specimens. CONCLUSION(S): The data demonstrate for the first time the occurrence of AAV infection in human semen. Sperm motility seems to be affected by the presence of AAV.

Infection with adeno-associated virus type 5 inhibits mutagenicity of herpes simplex virus type 1 or 4-nitroquinoline-1-oxide.

Infection with adeno-associated virus type 5 (AAV-5) reduced the number of mutants arising in the hypoxanthine phosphoribosyltransferase locus of human RD 176 cells after infection with herpes simplex virus type 1 (HSV-1; partially inactivated) or 4-nitroquinoline-1-oxide (4-NQO). The mutation frequency was reduced by AAV-5 infection from 11.4 to 1.8 after mutation with HSV-1 and from 3.2 to 2.5 when mutation was induced by 4-NQO. This was analyzed by determination of the number of cells resistant to 8-azaguanine when infected with AAV-5 prior to induction of mutations with HSV-1 or 4-NQO.

DNA amplification in genetic toxicology.

Chemical compounds can cause amplification of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile systems to study amplification of specific target genes in untransformed cells and in tumor cells.

Induction of selective DNA amplification and morphological cell transformation in Syrian hamster embryo cells.

DNA amplification is a frequently observed event in continuous cell lines and in tumors. It is likely that a common mechanism underlies the amplification of specific DNA sequences which confer drug resistance and genes which give a growth advantage to the tumor. To find a correlation between the induction of DNA amplification by chemicals and morphological cell transformation we treated Syrian hamster embryo (SHE) cells with diverse antineoplastic agents of different classes. Analysis of these agents seems to be important since they are potentially carcinogenic and resistance inducing. For the measurement of DNA amplification we established a new system using adeno-associated virus type 2 (AAV)-infected primary SHE cells as target cells and amplification of viral DNA as marker of DNA amplification. Simultaneously we determined morphological cell transformation in SHE cells. Our findings demonstrate that there is only a limited correlation between the induction of AAV DNA amplification and the morphological cell transformation in SHE cells. The newly established system of AAV DNA amplification appears to be a useful tool for the investigation of drug resistance in target cells of choice.

Detection of mutations in bacteria and of DNA damage and amplified DNA sequences in mammalian cells as a systematic test strategy for elucidating biological activities of chemical carcinogens.

The interdisciplinary evaluation of risks from carcinogens utilizes, inter alia, data on the activities of the compounds in short-term assays. A systematic approach is being used to determine mutagenesis in bacteria (the study of direct activities and specific modes of metabolic activation), DNA damage within primary mammalian cells (DNA single-strand breaks and persistence of damage, by a method extendable to the in vivo situation) and amplified DNA sequences in cultured cells (as an endpoint probably relevant to carcinogenesis). This test combination was expected to reduce some of the shortcomings of other batteries of tests, which suffer from a lack of appropriate metabolic conversion of compounds, irrelevancy of genetic endpoints and pharmacokinetic limitations. Furthermore, as each assay in the test strategy differs from the others only by one of the parameters described above, a reasonable understanding of divergent test results from assay to assay was anticipated. Several substances were investigated to elucidate why their activities in short-term assays and in carcinogenesis experiments do not correlate. The substances were N-nitrodimethylamine, for which formaldehyde is the reactive intermediate in bacterial mutagenesis but not in mammalian cells or in vivo, N-nitrosodiethanolamine, a carcinogen that must be activated by external alcohol dehydrogenase to be mutagenic in bacteria, N-nitrosodialkylamines, with unique organotropism in vivo for which organ-specific activation was studied in vitro, N-nitroso compounds that are inactivated in vivo but not in vitro, and components of the aristolochic acid mixture which may be metabolized oxidatively or reductively, as well as numerous miscellaneous compounds that were expected to be genotoxins on account of their chemical structure. In addition to the assessment of genotoxicity, the results obtained in individual tests of this strategy yield important data on mechanisms of activity, such as organ-specific activation and deactivation, species variations, in vitro/in vivo correlation and persistence or repair of damage.

[Natural infection with adeno-associated viruses]. / Infection naturalle à virus adéno-associés.

Adeno-associated viruses (AAV) are parvoviruses which exhibit oncosuppressive properties as well as unique characteristics of integration. They have never been associated with human diseases. AAV are thus considered promising vectors for the corrective therapy of various gene defects. In this review, the (possible) consequences of AAV infection for human health, cancer development and recombinant AAV vectors are discussed with respect to recent results on the cellular and molecular targets of AAV infection in humans.