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Chemical scoring system for hazard assessment
M O L E C U L A R P R O B E S TO D E T E C T P O I N T M U T A T I O N IN M A M M A L I A N SPERM
Sohaib A. Khan
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521
Occupational exposure of chemicals is a serious problem and can be responsible for a variety of toxic manifestations. A number of criteria are presently in use to monitor the population who are exposed to chemicals at work sites to d e t e r m i n e if a n y deleterious effect might have occurred. However, there is lack of a test t h a t can directly measure the effect of toxic chemicals at the genomic level in the exposed individual. The monitoring of mutation systems based on whole .animals requires a pool of large numbers of a n i m a l s t h a t seems impractical and expensive for routine testing of mutagens. As an alternative to monitoring the pedigree, cells from exposed individuals m a y be considered for screening of point mutations through the use of an appropriate molecular probe directed towards a specific locus. Mammalian spermatogenic cells, which differentiate within a physiological environment during mutagenic treatment, are ideally suited for the detection of mutation. By using recombinant DNA techniques, I am developing a probe for the lactate dehydrogenase-X (LDH-X) locus. Toward this end, a mouse testis cDNA library has been constructed in an expression vector, ~ g t l l . Results obtained concerning cloning of LDH-X cDNA, detection of LDH-X mRNA by Northern blot analysis, and in vitro translation data of mouse testis RNA are discussed. Furthermore, a strategy showing how the LDH-X probe can be used to detect point mutation at the LDH-X locus using RNA/DNA hybridization and in situ hybridization is presented.
C H E M I C A L S C O R I N G SYSTEM F O R H A Z A R D A S S E S S M E N T
Robert H. Ross, l* Terry O'Bryan,2 and Po-Yung Lu 1
I Chemical Hazard Evaluation Program, Information Research and Analysis Section, Biology Division,Oak Ridge National Laboratory, Oak Ridge, Tennessee 32830 and 2Existing Chemicals Assessment Division, U.S. Environmental Protection Agency, Washington, DC 20460
To assist in the preliminary e v a l u a t i o n of compounds of toxicological a n d environmental interest to the U.S. Environmental Protection Agency (US EPA), a
215
scoring system was devised as a collaborative effort between the US EPA and the Oak Ridge National Laboratory. The scoring system combines objective guidelines with professional judgment to evaluate chemicals and consists of 17 separate scoring parameters, seven of which pertain directly to toxicology, e.g., oncogenicity and genotoxicity. The remaining parameters are related to environmental fate and occupational and consumer exposure. This scoring system was designed to rapidly score chemicals in a minimal amount of time with readily available information. It is used by the Office of Toxic Substances of the US EPA as a tool to help set priorities in conjunction with other criteria. It is particularly useful in performing preliminary evaluations involving large chemical classes, such as petroleum distillates. Supported by the U.S. E n v i r o n m e n t a l Protection Agency under I n t e r a g e n c y Agreement No. DW89930405-01-2.
C A N E A R T H W O R M S S U B S T I T U T E F O R R O D E N T S IN D E T E R M I N I N G A C U T E M E T A L TOXICITY?
S. Serda a n d A r t h u r Furst*
Institute of Chemical Biology, University of San Francisco, San Francisca, California 941 l 7-1080
Earthworms have been known to accumulate heavy metals in their tissues. The posterior a l i m e n t a r y tract, specifically the c h l o r a g o g e n o u s tissue, a p p e a r s to concentrate most of the heavy metals (Ireland and Richards, 1977). The chloragogen tissue seems to function as the invertebrate's liver (Roots, 1960). The LDs0 values for metals can be determined by injecting the compounds into the hemocoel of the worms. This route is justified, for when worms were subjected to cadmium in soil, it was found that 68% of the cadmium was associated with the surface mucous (Flemming, 1981). The metal complexes formed from the interaction .between surface mucous and the metal are soluble and are not thought to interfere with the cellular activities of the organism. The LD50 values for the sodium salts of selenite and selenate have been identified. This procedure for LDs0 determination leads to reproducible toxicity data and may allow us to use earthworms as an alternative to the mammalian system.
216
Sohaib A. Khan
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521
Occupational exposure of chemicals is a serious problem and can be responsible for a variety of toxic manifestations. A number of criteria are presently in use to monitor the population who are exposed to chemicals at work sites to d e t e r m i n e if a n y deleterious effect might have occurred. However, there is lack of a test t h a t can directly measure the effect of toxic chemicals at the genomic level in the exposed individual. The monitoring of mutation systems based on whole .animals requires a pool of large numbers of a n i m a l s t h a t seems impractical and expensive for routine testing of mutagens. As an alternative to monitoring the pedigree, cells from exposed individuals m a y be considered for screening of point mutations through the use of an appropriate molecular probe directed towards a specific locus. Mammalian spermatogenic cells, which differentiate within a physiological environment during mutagenic treatment, are ideally suited for the detection of mutation. By using recombinant DNA techniques, I am developing a probe for the lactate dehydrogenase-X (LDH-X) locus. Toward this end, a mouse testis cDNA library has been constructed in an expression vector, ~ g t l l . Results obtained concerning cloning of LDH-X cDNA, detection of LDH-X mRNA by Northern blot analysis, and in vitro translation data of mouse testis RNA are discussed. Furthermore, a strategy showing how the LDH-X probe can be used to detect point mutation at the LDH-X locus using RNA/DNA hybridization and in situ hybridization is presented.
C H E M I C A L S C O R I N G SYSTEM F O R H A Z A R D A S S E S S M E N T
Robert H. Ross, l* Terry O'Bryan,2 and Po-Yung Lu 1
I Chemical Hazard Evaluation Program, Information Research and Analysis Section, Biology Division,Oak Ridge National Laboratory, Oak Ridge, Tennessee 32830 and 2Existing Chemicals Assessment Division, U.S. Environmental Protection Agency, Washington, DC 20460
To assist in the preliminary e v a l u a t i o n of compounds of toxicological a n d environmental interest to the U.S. Environmental Protection Agency (US EPA), a
215
scoring system was devised as a collaborative effort between the US EPA and the Oak Ridge National Laboratory. The scoring system combines objective guidelines with professional judgment to evaluate chemicals and consists of 17 separate scoring parameters, seven of which pertain directly to toxicology, e.g., oncogenicity and genotoxicity. The remaining parameters are related to environmental fate and occupational and consumer exposure. This scoring system was designed to rapidly score chemicals in a minimal amount of time with readily available information. It is used by the Office of Toxic Substances of the US EPA as a tool to help set priorities in conjunction with other criteria. It is particularly useful in performing preliminary evaluations involving large chemical classes, such as petroleum distillates. Supported by the U.S. E n v i r o n m e n t a l Protection Agency under I n t e r a g e n c y Agreement No. DW89930405-01-2.
C A N E A R T H W O R M S S U B S T I T U T E F O R R O D E N T S IN D E T E R M I N I N G A C U T E M E T A L TOXICITY?
S. Serda a n d A r t h u r Furst*
Institute of Chemical Biology, University of San Francisco, San Francisca, California 941 l 7-1080
Earthworms have been known to accumulate heavy metals in their tissues. The posterior a l i m e n t a r y tract, specifically the c h l o r a g o g e n o u s tissue, a p p e a r s to concentrate most of the heavy metals (Ireland and Richards, 1977). The chloragogen tissue seems to function as the invertebrate's liver (Roots, 1960). The LDs0 values for metals can be determined by injecting the compounds into the hemocoel of the worms. This route is justified, for when worms were subjected to cadmium in soil, it was found that 68% of the cadmium was associated with the surface mucous (Flemming, 1981). The metal complexes formed from the interaction .between surface mucous and the metal are soluble and are not thought to interfere with the cellular activities of the organism. The LD50 values for the sodium salts of selenite and selenate have been identified. This procedure for LDs0 determination leads to reproducible toxicity data and may allow us to use earthworms as an alternative to the mammalian system.
216