Biomarker correlations of urinary 2,4-D levels in foresters: genomic instability and endocrine disruption.

Forest pesticide applicators constitute a unique pesticide use group. Aerial, mechanical-ground, and focal weed control by application of herbicides, in particular chlorophenoxy herbicides, yield diverse exposure scenarios. In the present work, we analyzed aberrations in G-banded chromosomes, reproductive hormone levels, and polymerase chain reaction-based V(D)J rearrangement frequencies in applicators whose exposures were mostly limited to chlorophenoxy herbicides. Data from appliers where chlorophenoxy use was less frequent were also examined. The biomarker outcome data were compared to urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) obtained at the time of maximum 2,4-D use. Further comparisons of outcome data were made to the total volume of herbicides applied during the entire pesticide-use season.Twenty-four applicators and 15 minimally exposed foresters (control) subjects were studied. Categorized by applicator method, men who used a hand-held, backpack sprayer in their applications showed the highest average level (453.6 ppb) of 2,4-D in urine. Serum luteinizing hormone (LH) values were correlated with urinary 2,4-D levels, but follicle-stimulating hormone and free and total testosterone were not. At the height of the application season; 6/7 backpack sprayers, 3/4 applicators who used multinozzle mechanical (boom) sprayers, 4/8 aerial applicators, and 2/5 skidder-radiarc (closed cab) appliers had two or more V(D)J region rearrangements per microgram of DNA. Only 5 of 15 minimally exposed (control) foresters had two or more rearrangements, and 3 of these 5 subjects demonstrated detectable levels of 2,4-D in the urine. Only 8/24 DNA samples obtained from the exposed group 10 months or more after their last chlorophenoxy use had two rearrangements per microgram of DNA, suggesting that the exposure-related effects observed were reversible and temporary. Although urinary 2,4-D levels were not correlated with chromosome aberration frequency, chromosome aberration frequencies were correlated with the total volume of herbicides applied, including products other than 2,4-D. In summary, herbicide applicators with high urinary levels of 2,4-D (backpack and boom spray applications) exhibited elevated LH levels. They also exhibited altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible months after peak exposure. Though highly detailed, the limited sample size warrants cautious interpretation of the data.

Molecular evidence that the capacity for endosporulation is universal among phototrophic heliobacteria.

Although enrichment cultures for anoxygenic phototrophic heliobacteria commonly contain sporulating cells, once strains of heliobacteria are obtained in pure culture, they all but cease to sporulate. In fact, some species of heliobacteria have never been observed to sporulate. Thus, despite their phylogenetic connection to endospore-forming bacteria, the question of sporulation capacity in heliobacteria remains open. We have investigated this problem using PCR and Southern hybridization as tools and show here that all recognized species of heliobacteria tested, as well as several unclassified strains, contain homologs to the ssp genes of Clostridium and Bacillus species, genes that encode key sporulation-specific proteins. It can therefore be concluded that as a group, heliobacteria are likely all to be endospore-forming bacteria in agreement with their phylogenetic placement within the 'low GC' Gram-positive bacteria.

Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene.

Nerve growth factor (NGF) induces neurite outgrowth and promotes survival of embryonic sensory and sympathetic neurons in culture. In vivo, NGF decreases the extent of naturally occurring cell death in developing sympathetic ganglia and protects cholinergic neurons of the basal forebrain and caudatoputamen. NGF interacts with the low-affinity p75 receptor and with Trk, a receptor tyrosine kinase encoded by the trk proto-oncogene. To study the role of Trk in vivo, we have ablated the gene in embryonic stem cells by homologous recombination. Mice lacking Trk have severe sensory and sympathetic neuropathies and most die within one month of birth. They have extensive neuronal cell loss in trigeminal, sympathetic and dorsal root ganglia, as well as a decrease in the cholinergic basal forebrain projections to the hippocampus and cortex. These findings demonstrate that Trk is the primary mediator of the trophic actions of NGF in vivo and that this signalling pathway plays a crucial role in the development of both the peripheral and the central nervous systems.

Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death.

We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145trkB, a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkBTK(-/-), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkBTK (-/-) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp145trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system.

ras gene Amplification and malignant transformation.

Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.

The immediate-early enhancer element of herpes simplex virus type 1 can replace a regulatory region of the c-Ha-ras1 oncogene required for transformation.

A 0.8-kilobase SacI DNA fragment in the distal 5'-noncoding region of the c-Ha-ras1 oncogene hybridized to high guanine X cytosine sites of herpes simplex virus type 1 (HSV-1) DNA restriction fragments. Nucleotide sequence comparisons localized one of these sites to the intergenic region of HSV between the immediate-early genes coding for IEmRNA-3 and IEmRNA-4/5 that has enhancer-type activity. We tested the possibility that the HSV-1 enhancer and the upstream c-Ha-ras1 SacI fragment were functionally related by assaying for the capacity of recombinant plasmids in which the HSV-1 enhancer replaced the oncogene 0.8-kilobase SacI fragment to transform NIH/3T3 cells. Deletion of the 0.8-kilobase SacI fragment abolished the biological activity of c-Ha-ras1, but its replacement by the HSV-1 enhancer fully restored it. These results confirm the enhancer properties of the HSV-1 immediate-early intergenic region and suggest that c-Ha-ras1 sequences contained within the 0.8-kilobase SacI fragment plays a role in the transcriptional activation of the oncogene.

Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.

The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.

Transforming genes in human tumors.

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 X 10(4) focus forming units per picomole. These results indicate that we have molecularly cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcinoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.

Major structural proteins of type B, type C, and type D oncoviruses share interspecies antigenic determinants.

Interspecies antigenic determinants have been used as markers to study the evolution of oncoviruses. By radioimmunologic techniques, it has been possible to detect crossreactivities among the major structural proteins of prototype viruses representing each of the three major Oncovirinae genera. Two different subsets of interspecies antigenic determinants were demonstrated. One encompasses known type B and type D viruses; the other is shared by type D and mammalian type C viruses. Such determinants were also demonstrated in certain as yet unclassified oncoviruses. These findings provide experimental evidence supporting the concept of an evolutionary relationship among the three major oncovirus genera.

Natural immunity in mice to structural polypeptides of endogenous type C RNA viruses.

The immunological responses of inbred mice to structural components of one class of endogenous virus were investigated by means of radioimmunoassays utilizing highly purified viral proteins. Naturally occurring antiviral antibodies were demonstrated only in those strains possessing information for induction of a mouse cell-tropic endogenous virus. Moreover, these antibodies invariably appeared subsequent to the detection of spontaneous replication of this virus in the same animal. The immune responses elicited were much stronger against endogenous viral gp70 than p30, consistent with previous findings of tolerance in the mouse to the major structural antigen of its endogenous virus. However, the demonstration of an immune response to p30 under conditions of both natural and experimental immunization establishes that tolerance to this viral antigen can be overcome.