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Polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs) can enhance the generation of reactive oxygen species (ROS) by inducing cytochrome P450 1A (CYP 1A) in vivo and in vitro. While the brain is vulnerable to oxidative injury, whether or not CYP 1A induction in the brain can produce measurable levels of oxidative damage has not been reported. The objective of this study was to investigate the effect of this induction on oxidative damage to the CNS. Time course changes in rat brain CYP 1A activity were determined by measurement of ethoxyresorufin O-deethylase (EROD) activity in whole brain homogenates. Three days after exposure of rats to five daily injections of 3-methylcholanthrene (3-MC) an approximately sevenfold increase in EROD activity was observed. Hepatic levels were increased 60-100 fold. This increase in CYP 1A activity was not accompanied by increased protein or lipid oxidation as measured by tryptophan fluorescence and TBAR formation, or decreased glutamine synthetase (GS) activity. These findings indicate that if increased CYP 1A activity in the brain following 3-MC treatment leads to increased ROS generation, the increase is insufficient to overwhelm the endogenous antioxidant defense system, produce detectable oxidative damage, and alter glutamate homeostasis.

A rat cortical astrocyte preparation was used to investigate the effects of organotins on glutamate regulation by astrocytes. Exposure of astrocytes to low levels of organotins produced significant changes in two key components of glutamate homeostasis: glutamine synthetase (CS) activity and the high-affinity transport of L-glutamate. Trimethyltin (TMT), triethyltin (TET), and triphenyltin (TPT) exhibited differential abilities to reduce GS activity and glutamate uptake. Cultures incubated with 1 microM TET or TPT, but not TMT, exhibited a marked decrease in GS activity. Exposure to TET or TPT also produced a significant decrease in glutamate transport activity that was not observed with TMT. These declines in activity were not attributable to cell loss as measured by MTT reduction and lactate dehydrogenase (LDH) leakage. Since the loss of GS activity and transporter activity was not seen with acute organotin exposure, it is most likely attributable to a decreased presence of fully functioning protein. While the attenuation of GS and glutamate transporter activities by organotins does not match their pattern of neurotoxicity, the results indicate the potential for subtoxic concentrations of these compounds to increase extracellular glutamate and interact with other excitotoxic episodes.

The LRM55 rat astrocytoma cell line was used to study the time and concentration effects of trimethyltin (TMT) exposure on intracellular adenosine triphosphate (ATP) levels, formazan production from (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) release. TMT concentrations of > or =50 microM produced a delayed increase in extracellular LDH from approximately 20% at 24 h to almost 70%, at 72 h. Twenty-four hours before cell lysis was detectable ATP levels decreased to less than 30% and formazan production declined to 70% (50 microM), 31% (100 microM), and 21% (200 microM) of control values. Concentrations of TMT (5 and 10 microM) that produced little or no LDH release also decreased ATP levels (62 and 49% of control, respectively) and formazan production (63 and 52% of control, respectively) by 48 h. These data support the hypothesis that TMT exposure interferes with energy production and that this event likely contributes to the delayed cell death seen in this cell line. Moreover, the declines in ATP and formazan production can occur at subtoxic concentrations in LRM55 cells.

On the basis of reports that astrocytes play an important role in the neurotoxicity of trimethyltin (TMT), we investigated the sensitivity of astrocytes to TMT and compared it to triethyltin (TET), a neurotoxic analog with a different in vivo specificity. The gliotoxicity of these two compounds was further compared to that of tributyltin (TBT) and triphenyltin (TPT), two purportedly nonneurotoxic organotin compounds. The time and concentration components of organotin toxicity were determined by measuring lactate dehydrogenase (LDH) release and formazan production from dimethylthiazolyldiphenyltetrazolium bromide (MTT). A TMT concentration of 100 micromol/L did not elevate extracellular LDH until 48 h after exposure, while signs of toxicity were not seen at 72 h for concentrations less than 10 micromol/L. Extracellular LDH activity increased 24 h after exposure to concentrations of TET, TBT, and TPT as low as 2.5 micromol/L. TMT was the only organotin to produce a delayed cytotoxicity, requiring both higher concentrations and more time to produce discernible toxicity. In contrast with TBT and TPT, the toxicity of the two neurotoxic organotins (TMT and TET) produced an early increase in MTT reduction. The distinct pattern of toxicity for TMT does not explain its selective in vivo toxicity, but the lack of sensitivity of astrocytes to this organotin also does not rule out more subtle changes in these cells that could disrupt normal glial/neuronal interactions.

The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5-20 microM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 microM). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

The effect of potassium cyanide-induced chemical hypoxia on protein kinase C (PKC) translocation and cell injury was studied in differentiated PC12 cells. The cellular distribution of PKC in control cells and cells exposed to 100 microM and 1 mM KCN for 30 min. was visualized by use of an anti-PKC antibody and confocal laser scanning microscope. In control differentiated PC12 cells, PKC was localized perinuclearly, while following 12-phorbol 13-myristate acetate (PMA) or KCN it was translocated to the plasma and organelle membranes. Western blot analysis was used to quantify the translocation. Chemical hypoxia increased the membrane-bound PKC to 210% of control levels, while chelerythrine, a PKC inhibitor, and block of calcium influx into the cells (with calcium channel blocker and calcium-free medium) prevented this effect. Cyanide-induced PKC translocation persisted for at least 120 min. Cell injury was monitored by measuring lactate dehydrogenase (LDH) efflux from the cells 24 hr after addition of cyanide. PKC activation plays a role in hypoxic damage, since PKC down-regulation (by overnight exposure to PMA) or inhibition (with chelerythrine or staurosporine) conferred protection against KCN-induced cytotoxicity. Ca2+ channel blocker nifedipine also protected against chemical hypoxia. None of the pretreatments rendered complete protection against cyanide-induced hypoxia, indicating that PKC-independent mechanism(s) are also activated during chemical hypoxia and contribute to cell injury.

Protective effects of a perfluorooctylbromide emulsion on myocardial ischemia and reperfusion (MI/R) injury were evaluated in a modified Langendorff rat heart preparation. Isolated rat hearts were equilibrated in Krebs-Henseleit solution (KH) for 35 minutes and perfused with either cardioplegic solution (CPS) or a 100% perfluorooctylbromide (PFOB) emulsion in CPS for 3 minutes. Hearts were then bathed in the emulsion or CPS. Both groups were subjected to 30 minutes of ischemia. Following 30 minutes of ischemia and 30 minutes of reperfusion with KH solution, hearts subjected to the 100% PFOB emulsion showed improved recovery of left ventricular function. Tissue activities of the antioxidant enzymes glutathione peroxidase, superoxide dismutase, and catalase were not affected by the emulsion in this model. Activity of lactate dehydrogenase (LDH) in the bathing medium was elevated at the end of the experimental period in both control and PFOB-treated hearts. The PFOB emulsion reduced the decline in ATP and GSH levels produced by cardioplegia and subsequent reperfusion. No differences were noted in oxidized glutathione (GSSG) levels. These data suggest that the PFOB emulsion provides some protection for the myocardium against injury associated with cardioplegia.

The isolated, electrically-driven, guinea pig left atrium was used to study the ability of two perfluorocarbon emulsions to prevent anaerobic hypofunction in the myocardium. A 20% perfluorodecalin (PFD) emulsion maintained peak tension at 80% of aerated levels for more than 20 minutes. Emulsions of perfluorooctylbromide (PFOB) ranging from 25 to 100% produced a similar result, except that the 25% emulsion could not maintain contractions for the entire time period. Peak tension of atria bathed in K-H solution decreased to less than 50% over the same time period. Both 20% PFD and 100% PFOB maintained myocardial ATP levels at pre-hypoxic levels for at least twenty minutes after aeration was terminated. Unaerated atria, bathed in Krebs-Henseleit solution only, exhibited a significant decline in tissue ATP levels at this time. It appears that perfluorocarbon emulsions may delay oxygen desaturation and thereby protect cardiac tissue from ATP depletion and impaired cardiac function associated with hypoxia. This tissue preparation was found to be very useful for determining the efficacy of potential oxygen carriers.

Dibutyryl cyclic AMP and cyclic AMP in a DMSO medium were administered to the fetal rat heart in organ culture. Changes in heart rate were observed. Dibutyryl cyclic AMP in concentrations of 5 X 10(-5) M and 1 X 10(-4) M produced significant negative chronotropism. At a concentration of 1 X 10(-3) M, cyclic AMP in a 3% or 0.5% DMSO medium produced significant negative chronotropism. These data were compared with other studies. The negative chronotropic action of cyclic AMP and its dibutyryl derivative are evidence against the involvement of cyclic AMP in positive cardiac chronotropism.

The fetal rat heart in organ culture was used to investigate rate chanes by various cardioactive agents. Concomitantly, the effect of these pharmacologically induced rate changes on steady-state cyclic adenosine monophosphate levels was determined. Isoproterenol increased the fetal rat heart rate and cyclic adenosine monophosphate in a concentration-related fashion. Quinidine produced a concentration-related decrease in heart rate and no change in cyclic adenosine monophosphate level. The dysrhythmogenic agent produced concentration-related negative chronotropism in the fetal rat heart preparation and significant elevations in cyclic adenosine monophosphate at concentrations without chronotropic action. No correlation between chronotropic effect of a drug and cyclic adenosine monophosphate levels was observed.

The fetal rat heart in organ culture was used to study the interaction of calcium with the cardioactive agents. McN-2165 is an agent that produces negative chronotropism and elevation in cyclic AMP levels in this preparation. A concentration of 2.76 mM calcium ion antagonized the negative chronotropic action and prevented the cyclic AMP elevation produced by this agent. Positive chronotropic actions of isoproterenol were prevented by decreasing the calcium level below 1.84 mM.