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Residual damage to hematopoietic system in mice exposed to groundwater contaminants
brain regions affected by sublethal doses of cyanide, male rats were given sodium cyanide (NaCN) by controlled intravenous (iv) infusion (20 p l / m i n ; 5 m g / m l ) into the femoral vein. The infusion was temporarily halted when the rat lost its righting reflex and infusion .resumed when the righting reflex recurred. A pulse of [14C]2-deoxyglucose (2-DG) was given iv approximately 1 min or 60 rain after the N a C N infusion was started. In another group of rats, NaCN (0.3 pg) was given by intracerebral ventricular (icy) injection and 2-DG was given 5 rain later. Forty-five minutes -after the 2-DG pulse was given the rat was anesthetized with halothane, decapitated, the brain frozen in freon ( - 7 0 ° C ) , sectioned (20 lain) and the sections placed on rain-R-X-ray film for 21 days. Local cerebral glucose use (LCGU) in pmoles/q00g tissue/rain was measured in 42 brain regions. Results from selected brain regions are ~ v e n in Table 1. Table 1 LCGU (/tmoles/100 g/rain) in selected brain regions. Structure
Control
iv NaCN LCGU (1 min)
iv NaCN LCGU (60 min)
icv NaCN LCGU (5 min)
Frontalcortex Dorsalcaudate Corpuscallosum Globus paUidus Choroid plexus Substantia Nigra Ret.
113+7 124+5 33_+6 60 + 5 88-+ 5 69 5:6
101+ 9 126_+13 85_+ 6 * 89 + 8 * 158 4- 9 * 10b-+ 9 *
32+6 * 26_+9 * 12_+4 * 17-+ 4 * 80_+8 36 4- 2 *
49+ 4 * 57_+ 3 * 14± 2 * 26-+ 2 * 95 _+18 41 _+ 4 *
* P < 0.05 with respect to control Immediately after iv infusion of N a C N there is a significant increase in glucose use in only 5 out of 42 structures measured. These were: corpus callosum (2585;); lateral septum (1355;); bed nucleus striatum (1465;) choroid plexus (1805;) and substantia Idgra reticularis (1455;). Su~risingly, 60 rain after the initiation of N a C N infusion, L C G U was markedly suppressed in all brain regions except choroid plexus. Many grey matter regions (e.g., frontal cortex, cingulate, medial geniculate) have L C G U rates less than 305; of control. Likewise, L C G U was markedly suppressed 5 mh-l "~ter N a C N was given icy. Since the usual metabolic response to low energy levels in tissues is an increase in anaerobic glycolysis, a large increase in glucose use would be expected because glycolysis is less efficient than oxidative metabolism in the production of ATP. The marked reduction in glucose use after cyanide given by infusion or icv suggests that cyanide directly or indirectly has a strong depressant action on brain metabolism. An important question that remains to be answered is: how does cyanide suppress brain glucose use? Indirectly, is there a compensation mechanism that reduces brain function, thus lewering the rate of energy use when energy supply is below a critical level? or does cyanide have a direct suppressive effect on brain function? This work was supported by D A M D 17-86-G-6038.
P.we.445] Residual damage to hematopoiefic system in mice exposed to groundwater contaminants H o n g , H.L., Y a n g , R . S . H . a n d B o o r m a n ,
G,A.
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709, U.S.A.
To assess the potential health effects of chemically contaminated groundwater, we initiated a toxicology program on a mixture of 25 frequently detected groundwater contaminants derived from hazardous waste disposal sites. In order to minimize chemical interactions, the stock solution was stored as 2 substocks solutions: an organic substock containing 19 organic chemicals and an aqueous substock containing 6 metals and phenol. Fresh drinking water mixture was prepared from substocks every 48-72 hours aud analyzed penodicaUy both before and after dosing. As part of this toxicology program, myelotoxicity studies were conducted. Evidence has accumulated that exposure to several environmental pollutants of this chemical mixture, such as halogenated aromatic hydrocarbons, polycyclic
1543 aromatics, and heavy metals, can produce myelotoxicity in laboratory animals at dose levels without other manifestations of toxicity. Bone marrow with rapidly renewing populations is one of the most sensitive tissues to cytotoxic agents. In humans, bone marrow failure is a significant complication of cancer chemotherapy and has been implicated in exposures to drugs and environmental agents. Further, mice exposed to a myelotoxic agent shortly after birth were more sensitive to irradiation 5 months later (Hong et al., 1988). In the present study, bone marrow parameters were examined in mice exposed to 0, 1, 5 or 10~ of a simulated chemical mixture stock solution of groundwater contaminants in drinking water for 15.5 weeks. The possible toxicological interaction of groundwater contaminants and irradiation of hematopoiesis was investigated by determining proliferation of marrow granulocyte macrophage progenitors (CFU-GM). Mice treated with 5 and 10~ of chemical mixture stock for 15.5 weeks showed suppressed CFU-GM; however, this suppression returned to normal values in 10 weeks following the cessation of treatment. If, on the other hand, mice were received with 200 rads whole body irradiation at 2 and 9 weeks during this 10-week recovery period, the combined treatment (i.e., chemical mixture followed by irradiation) resulted in a significantly slower recovery of CFU-GMs as compared to irradiation alone. This study showed that after cessation of chemical mixture treatment when hematological parameters were normal, a residual effect of the chemical mixture could be demonstrated following irradiation~ Thuso re.joe expose,J to ~oundwater contaminants for 15.5 weeks are more sensitive to subsequent irradiation-induced injury. It seems that the toxicology of chemical mixtures of environmental levels will probably not involve acute toxic responses. It is most likely an insidious effect disrupting the homeostasis of the organism. Based on conventional toxicological endpomts, the animal may appear clinically normal; however, final toxicity may be expressed by the enhancement or potentiation of other exposure of chemical, physical, a n d / o r biological agents. This is important to people living in areas where the potential for multiple chemical exposure at higher than normal levels is great. Therefore, long-term exposure to highly contaminated groundwater represents a potential risk to the hematopoietic system. Reference Hong, H.L., Silver, M., and Boorman, G.A., 1988, Demonstration of residual marrow effect in mice exposed to ethylene glycol monomethyl ether. Toxicology, 50, 107-115.
I P.we.446 ]
Cytoplasmic vacuolation induced by bis-tertiary aikyl derivatives Valtolina, M., and Forster, R. Italfarmaco Research Centre, via dei Lavoratori 54, 20092 Cinisello Balsamo, Milano, Italy
The induction of cytoplasmic vacuolation by derivatives of alkyl-bis-tertiary-amines has been studied in cultured cells. Tetramethyl-l,4-butanediamine and-Tetramethyl-l,6-hexanediamine were used as model compounds; intracellular vacuolation caused by these coml: .... ,is in RBT CC-8 rat urinary bladder carcinoma cells and primary rabbit aorta smooth muscle cells has previously been reported by Rorig et al. (1987). We examined the uptake of these products into an embryo-derived intestinal epithelial cell line (l 407) and in primary human smooth muscle cells, as models of the potential target tissues after oral exposure. In order to observe uptake, the culture medium was maintained alkaline (at pH 8.00, simulating conditions in the small intestine); the alkaline pH was essential for the observation of vacuolation, probably through the reduction of ionisation of the tertiary amine groups. Marked induction of intracellular vacuolation was observed after as little as two hours treatment, and the phenomenon was fully expressed by 24 hours of treatment. Extremely numerous small vacuoles were observed in the cytoplasm surrounding the nucleus. The effect was observed over a concentration range from 30 uM to 10 mM at greater concentrations the products became cytotoxic, resulting in the detachment of the cells. Fixation and staining of the cells allowed the elaboration of a visual scoring scheme; quantitative studies using image analysis are currently in course. On removal of the treatment medium, the vacuoles disappeared, the cells regained a normal appearance and continued to divide and grow. After 48 hours in fresh medium recovery appeared to be full, suggesting that the phenomenon is reversible. The relevance of our findings to the sequelae of in vivo treatment with the two model compounds is currently under study.
Control
iv NaCN LCGU (1 min)
iv NaCN LCGU (60 min)
icv NaCN LCGU (5 min)
Frontalcortex Dorsalcaudate Corpuscallosum Globus paUidus Choroid plexus Substantia Nigra Ret.
113+7 124+5 33_+6 60 + 5 88-+ 5 69 5:6
101+ 9 126_+13 85_+ 6 * 89 + 8 * 158 4- 9 * 10b-+ 9 *
32+6 * 26_+9 * 12_+4 * 17-+ 4 * 80_+8 36 4- 2 *
49+ 4 * 57_+ 3 * 14± 2 * 26-+ 2 * 95 _+18 41 _+ 4 *
* P < 0.05 with respect to control Immediately after iv infusion of N a C N there is a significant increase in glucose use in only 5 out of 42 structures measured. These were: corpus callosum (2585;); lateral septum (1355;); bed nucleus striatum (1465;) choroid plexus (1805;) and substantia Idgra reticularis (1455;). Su~risingly, 60 rain after the initiation of N a C N infusion, L C G U was markedly suppressed in all brain regions except choroid plexus. Many grey matter regions (e.g., frontal cortex, cingulate, medial geniculate) have L C G U rates less than 305; of control. Likewise, L C G U was markedly suppressed 5 mh-l "~ter N a C N was given icy. Since the usual metabolic response to low energy levels in tissues is an increase in anaerobic glycolysis, a large increase in glucose use would be expected because glycolysis is less efficient than oxidative metabolism in the production of ATP. The marked reduction in glucose use after cyanide given by infusion or icv suggests that cyanide directly or indirectly has a strong depressant action on brain metabolism. An important question that remains to be answered is: how does cyanide suppress brain glucose use? Indirectly, is there a compensation mechanism that reduces brain function, thus lewering the rate of energy use when energy supply is below a critical level? or does cyanide have a direct suppressive effect on brain function? This work was supported by D A M D 17-86-G-6038.
P.we.445] Residual damage to hematopoiefic system in mice exposed to groundwater contaminants H o n g , H.L., Y a n g , R . S . H . a n d B o o r m a n ,
G,A.
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709, U.S.A.
To assess the potential health effects of chemically contaminated groundwater, we initiated a toxicology program on a mixture of 25 frequently detected groundwater contaminants derived from hazardous waste disposal sites. In order to minimize chemical interactions, the stock solution was stored as 2 substocks solutions: an organic substock containing 19 organic chemicals and an aqueous substock containing 6 metals and phenol. Fresh drinking water mixture was prepared from substocks every 48-72 hours aud analyzed penodicaUy both before and after dosing. As part of this toxicology program, myelotoxicity studies were conducted. Evidence has accumulated that exposure to several environmental pollutants of this chemical mixture, such as halogenated aromatic hydrocarbons, polycyclic
1543 aromatics, and heavy metals, can produce myelotoxicity in laboratory animals at dose levels without other manifestations of toxicity. Bone marrow with rapidly renewing populations is one of the most sensitive tissues to cytotoxic agents. In humans, bone marrow failure is a significant complication of cancer chemotherapy and has been implicated in exposures to drugs and environmental agents. Further, mice exposed to a myelotoxic agent shortly after birth were more sensitive to irradiation 5 months later (Hong et al., 1988). In the present study, bone marrow parameters were examined in mice exposed to 0, 1, 5 or 10~ of a simulated chemical mixture stock solution of groundwater contaminants in drinking water for 15.5 weeks. The possible toxicological interaction of groundwater contaminants and irradiation of hematopoiesis was investigated by determining proliferation of marrow granulocyte macrophage progenitors (CFU-GM). Mice treated with 5 and 10~ of chemical mixture stock for 15.5 weeks showed suppressed CFU-GM; however, this suppression returned to normal values in 10 weeks following the cessation of treatment. If, on the other hand, mice were received with 200 rads whole body irradiation at 2 and 9 weeks during this 10-week recovery period, the combined treatment (i.e., chemical mixture followed by irradiation) resulted in a significantly slower recovery of CFU-GMs as compared to irradiation alone. This study showed that after cessation of chemical mixture treatment when hematological parameters were normal, a residual effect of the chemical mixture could be demonstrated following irradiation~ Thuso re.joe expose,J to ~oundwater contaminants for 15.5 weeks are more sensitive to subsequent irradiation-induced injury. It seems that the toxicology of chemical mixtures of environmental levels will probably not involve acute toxic responses. It is most likely an insidious effect disrupting the homeostasis of the organism. Based on conventional toxicological endpomts, the animal may appear clinically normal; however, final toxicity may be expressed by the enhancement or potentiation of other exposure of chemical, physical, a n d / o r biological agents. This is important to people living in areas where the potential for multiple chemical exposure at higher than normal levels is great. Therefore, long-term exposure to highly contaminated groundwater represents a potential risk to the hematopoietic system. Reference Hong, H.L., Silver, M., and Boorman, G.A., 1988, Demonstration of residual marrow effect in mice exposed to ethylene glycol monomethyl ether. Toxicology, 50, 107-115.
I P.we.446 ]
Cytoplasmic vacuolation induced by bis-tertiary aikyl derivatives Valtolina, M., and Forster, R. Italfarmaco Research Centre, via dei Lavoratori 54, 20092 Cinisello Balsamo, Milano, Italy
The induction of cytoplasmic vacuolation by derivatives of alkyl-bis-tertiary-amines has been studied in cultured cells. Tetramethyl-l,4-butanediamine and-Tetramethyl-l,6-hexanediamine were used as model compounds; intracellular vacuolation caused by these coml: .... ,is in RBT CC-8 rat urinary bladder carcinoma cells and primary rabbit aorta smooth muscle cells has previously been reported by Rorig et al. (1987). We examined the uptake of these products into an embryo-derived intestinal epithelial cell line (l 407) and in primary human smooth muscle cells, as models of the potential target tissues after oral exposure. In order to observe uptake, the culture medium was maintained alkaline (at pH 8.00, simulating conditions in the small intestine); the alkaline pH was essential for the observation of vacuolation, probably through the reduction of ionisation of the tertiary amine groups. Marked induction of intracellular vacuolation was observed after as little as two hours treatment, and the phenomenon was fully expressed by 24 hours of treatment. Extremely numerous small vacuoles were observed in the cytoplasm surrounding the nucleus. The effect was observed over a concentration range from 30 uM to 10 mM at greater concentrations the products became cytotoxic, resulting in the detachment of the cells. Fixation and staining of the cells allowed the elaboration of a visual scoring scheme; quantitative studies using image analysis are currently in course. On removal of the treatment medium, the vacuoles disappeared, the cells regained a normal appearance and continued to divide and grow. After 48 hours in fresh medium recovery appeared to be full, suggesting that the phenomenon is reversible. The relevance of our findings to the sequelae of in vivo treatment with the two model compounds is currently under study.