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Changes in the metabolic responses of brain tissue to stimulation, in vitro, produced by in vivo administration of methyl mercury
Neuropharmacology.
1975, 14. 351-359. Pergamon
Press.
Printed
in Gt. Britain.
CHANGES IN THE METABOLIC RESPONSES OF BRAIN TISSUE TO STIMULATION, IN VITRO, PRODUCED BY IN VII/O ADMINISTRATION OF METHYL MERCURY R. J. BULL and S. D. LUTKENHOFF Water Supply Research Laboratory, National Environmental Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 (Accepted
11September 1974)
Summary~Electrical stimulation of rat cerebral cortex slices produced responses in the redox state of nicotinamide-adenine dinucleotides (NAD(P))* and cytochrome intermediates which were measured spectrophotometrically. These responses could be divided into three distinct phases: (I) an initial oxidative phase, followed by (2) a reductive phase and (3) a relaxation to the prestimulation baseline. Adult SpragueeDawley rats were exposed to intraperitoneal injections of methyl mercuric chloride in doses ranging from 0.005 to 2.0 mg/kg per day for a period of 14 days. Methyl mercury enhanced the reductive response at low doses and inhibited at higher doses. At doses of 0.05 mg/kg per day and higher, methyl mercury significantly inhibited the reoxidation of NAD(P) reduced by electrical stimulation. An increase in the media potassium concentration from 3 to 30 mM resulted in an initial oxidation followed by a net reduction of NAD(P). Inhibition of the initial oxidative phase of the response to potassium stimulation became evident at doses above 0.15 mg/kg per day. The reductive response of the NAD(P) to addition of potassium was also enhanced by low doses of methyl mercury. In contrast to results with electrical stimulation, inhibition of this response at high doses was not as apparent. However, aerobic glycolytic rate in response to addition of potassium was found to be inhibited at 2.0 mg/kg per day. These results are discussed in terms of the necessary integration of energy metabolism with the function of brain.
The occurrence of methyl mercury in the aquatic environment has been extensively documented in recent years (see, e.g. JENSEN and JERNELOV, 1969). Mercury in this form has been shown to gain access to the food chain of man (WESTOO,1969, RIVERS, PEARSON and SHULTZ, 1972). These findings have stimulated interest in the toxicological properties peculiar to alkylated derivatives of mercury. Although it is generally agreed that the nervous system is predominately involved in the toxicity of the alkyl mercurials, there is little information dealing with methyl mercury-induced changes in the biochemistry and physiology of the central nervous system that are related to a gradation of exposures over a time period. Data derived from various investigations (see, e.g. CLARKSON, 1972) have documented important differences in the physiological availability and distribution of these compounds relative to the inorganic salts of mercury. As a consequence, considerable emphasis has been placed on these differences to account for the clearly different toxic symptoms associated with the alkyl mercurials. Although one can show a relative increase of mercury in the central nervous system when methyl mercury, as opposed to the inorganic mercurials, is administered to animals, the brain still remains low in mercury concentration relative to liver and kidney with either acute or chronic exposures (FRIBERG, 1971). While this does not negate the importance of the distributional differences as being partially responsible for the predominance of nervous system symptoms with the alkyl mercurials, it is apparent that the effects are also a result of a metabolism peculiar to nerve tissue. One way in which brain metabolism has long been recognized as unique, is that adequate amounts of glucose and oxygen must be supplied continuously to avoid compromise of
* The abbreviation NAD(P) is used to designate both forms of the nicotinamide-adenine dinucleotides (i.e. NAD and NADP) because spectral differentiation of the compounds is not possible. In brain, the changes observed are most likely ascribed to NAD for a variety of reasons, but this cannot be regarded as proven. 351
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R. J. BULL andS. D. LUTKENHOFF
function (SHALIT,BELLERand FEINSOD,1972). Previous reports have indicated that methyl mercury produces alterations in glucose metabolism in the brain of rats (PATERSONand USHER, 197 1; YOSHINO,MOZAI and NAKAO, 1966). On the basis of these earlier reports, we decided to examine the relationship between the functional activity and energy metabolism in slices of cerebral cortex taken from rats exposed to methyl mercury. Our approach depends on the kinetics of changes induced in various metabolic parameters in the transition from a resting to an active state (depolarization). Methyl mercury has been found to produce rather complex changes in these metabolic responses suggesting that the coordination of energy metabolism to functional activity (i.e. metabolic control) has been impaired. METHODS
Treatment of’animals Adult Sprague-Dawley rats (450-600 g) were injected intraperitoneally with methyl mercuric chloride, dissolved in distilled water, at doses ranging from 0.005 to 2-Omg/kg per day as the salt (20% less as Hg) for a 14-day period. Control animals were injected with an equal volume of distilled water. All solutions were adjusted to a pH of 5.5. Food and water were supplied ad lib. On the fifteenth day the animals were sacrificed. Preparation and incubation o~tissues Brain slices were prepared from the surface of each hemisphere of the cerebral cortex using a Stadie-Riggs microtome blade and glass guide (BULL and LUTKENHOFF,1973). Slices used for measurement of oxygen uptake and aerobic glycolytic rate were touched to a clean glass surface to remove adhering media and were weighed to the nearest 05 mg. Determination of the cross-sectional area and weight of eight brain slices was used to calculate their average thickness, assuming equal specific gravity with water. The figure obtained was 0.41 3_ 0.02 mm (S.E.M.). In all cases, the incubation media initially consisted of (MM); NaCl, 127; Mg&, 1.3; NaH,PO1, l-3; CaCl,, 0.75; NaHCOs, 26; and KCl, 3.0. Where indicated, 27 mM KC1 was added to bring the media potassium concentration to 30 mM. The incubation media was continuously oxygenated with 95% oxygen plus 5% carbon dioxide, thus maintaining pH at 7~4.Temperature of the incubation media was maintained at 37.0 f 0.2”C in both the spectrophotometric cuvette and the respirometer. The time course of each experiment consisted of a 30-min acclimatization period prior to the beginning of stimulation. At that point either the sequence of measurements of the responses to electrical stimulation was begun or potassium was added to stimulate the tissues. Experiments using electrical stimuIation lasted about one additional hour, whereas those utilizing potassium stimulation lasted for only another 20 min. In both cases, this was well within the survival time of the tissues, since preliminary experiments had indicated that the responses to electrical stimulation were mainlined without decrement for more than 2 hr. Absorbance of the ix-band of the cytochromes is also well-maintained over a similar time interval (BULL and CUMMINS,1973). Measurement of metabolic responses Spectrophotometric measurement of the responses of the respiratory intermediates of brain slices has been previously reported (CUMMINSand BULL, 1971; BULL and LUTKENHOFF,1973) and is based upon the pioneering work of Chance and associates in this area over the last 20 yr (see CHANCEand HOLLUNGER,1963). The responses to electrical stimulation were measured in each slice and in the following sequence 605-630 nm (cytochrome a), 562-575 nm (cytochrome b), 550-540 nm (cytochromes c + c,), 465-510 nm (flavoproteins), 445-455 nm (cytochrome a3) and 340-374 nm (nicotinamide-adenine dinucleotides, NAD(P)H). Electric field stimulation, consisting of biphasic square wave pulses of 7 V amplitude, 60 Hz, 0.5 msec pulse duration, was applied through enamelled silver wire electrodes. A Grass S88 stimulator, equipped with two stimulus isolation units, was used to generate the pulses. Pulse characteristics were continuously monitored on a cath-
Methyl mercury and the responses of brain
353
ode ray tube. The standard stimulus was a train of 10-Oset duration. To answer the question of how reproducible the responses to electrical stimulation might be, three separate control groups of six animals each were utilized. No significant differences were noted between the three groups of animals. Consequently, results were combined and are treated here as a single group. A similar operation was used with the potassium stimulated responses, but only two groups of six animals each were utilized. Each experimental group had six animals for studying the effects of electrically stimulated responses. In the case of potassium stimulation, the numbers utilized in each group involved a minimum of six animals per group for each parameter examined (i.e. not all parameters were looked at with each exposed animal). Cortex slices from methyl mercury exposed animals were also stimulated by increasing the media potassium concentration to 30 mM. The spectrophotometric, respiratory, and glycolytic responses to such stimulation have been previously described (BULL and LUTKENHOFF, 1973). The respiratory response of brain slices to addition of potassium was measured polarographically in a perfusion system in parallel experiments. Glycolytic rate of the tissues was followed by sampling the effluent of this latter apparatus at 2-min intervals and enzymatically assaying for lactic acid according to the method of HOHORST (1963). Mercury
determination
The mercury gradient observed in the cerebral cortex of rats exposed under the above conditions was determined in a separate experiment. Three groups of five rats were injected with 0.02, 0.2 and 2 mg/kg per day for 14 days. A fourth group was injected with distilled water over the same time period and served as control. Upon sacrifice (on 15th day) the brain was removed, subcortical matter dissected away, and cleaned of blood vessels with a cotton swab moistened with incubation media. Each hemisphere of the cerebral cortex (400-500mg) served as a sample. The tissues were digested in concentrated sulphuric (30 ml) and nitric acids (5 ml) and 2% potassium persulphate (10 ml) for 16 hr at 60°C. Mercury content was measured using flameless atomic absorption spectrophotometry. Average recovery was found to be 103%. RESULTS
As previously reported by other investigators (see, e.g. KLEIN, HERMAN, BRUBAKER and LUCIER, 1972), methyl mercury in sufficiently high doses, produced substantial losses in body weight. Consequently, animals on the 14&y intraperitoneal schedule of methyl mercury were weighed daily to get some measure of the gross toxicity of each dosage regimen. At 05 mg/kg per day, a significant decrease in the weight gained over this period was observed and at 2.0 mg/kg per day substantial losses in weight were recorded. Doses lower than 0.5 mg/kg per day had no observable effect on body weight. Table 1 indicates the concentrations of mercury reached in the cerebral cortex of animals dosed with methyl mercuric chloride in the range used in the present study. Mercury content of control animals was found to be quite low, barely greater than reagent blank values. Significant elevations in the level of mercury were found at 0.02 mg/kg per day. Higher doses progressively increased the mercury content of the cerebral cortex. Figure 1 illustrates the basic features of a response of the respiratory intermediates of cortical slices to electrical stimulation. The response pictured was recorded at 34&374 nm corresponding to NAD(P)H.Thisresponseand those measured at the other wavelength pairs Table 1. Accumulation of mercury in the cerebral cortex of rats given intraperitoneal injections of methyl mercuric chloride Dose of methyl mercuric chloride mg/kg per day x 14 0 0.02 0.2 2.0
Concentration of mercury in cerebral cortex (pg/g) 0.0049 0.0258 0.778 9.88
f + f f
Values represent the mean of five animals f S.E.M.
0+)020 O%I44 0.057 0.87
354
R.J. BULL and S.D. LUTKENHOFF
II I Phase
I
1
1
I2 1
I 3
1
Ttme
1
1
1
I
(min)
Fig. 1. Normal response of brain slices to a IO-set period of electrical stimulation illustrating its three phases. Measuring wavelengths, 340-374 nm.
can be divided into three phases for purposes of quantification; an initial oxidative phase, followed by a substantial reductive phase and a third phase of relaxation of the response back to the prestimulation baseline. For the purposes of this paper, data will be presented on the second and third phases. The initial oxidation was too rapid and variable for accurate characterization with the available instrumentation. The remainder of the response can be adequately described by two measurements. (1) An initial rate of reduction, which was measured as the slope of the second phase relative to baseline, and (2) a relaxation time of the response, describing the reoxidation of intermediate to prestimulation levels. Reoxidation of the NAD(P) reduced by electrical stimulation (Phase 3) was significantly slowed with doses of methyl mercuric chloride as low as 0.05 mg/kg per day (Fig. 2). This effect appeared to be maximal at 0.5 mg/kg per day, but was complicated at 2.0 mg/kg per day by a loss of the normal net reduction of NAD(P) in response to electrical stimulation in three of six animals. Instead, the responses were purely oxidative in direction relative to the prestimulation baseline, and allowed no measurement of the time constant to be made. The value indicated in Figure 2 for the 2.0 mg/kg per day dose was the average of the remaining three tissues. This effect appeared specific for the NAD(P) response. Only at 2 mg/kg per day was any alteration observed in the reoxidation of the cytochrome intermediates. Even these results were inconsistent and only in the case of cytochrome a (605630 nm) was the change statistically significant (i.e. increased from 47 to 85 set; P < 0.05). Lower doses of methyl mercuric chloride (0.01 mg/kg per day) were seen to substantially increase the rate of NAD(P) reduction with electrical stimulation (Fig. 3), but higher doses ( > 0.25 mg/kg per day) progressively decreased the rate of NAD(P) reduction (Phase 2) seen with electrical stimulation. Similar trends were observed in the cytochrome chain, i.e. a tendency for a more rapid reduction of intermediate at low doses followed by an apparent inhibition of this phase at higher doses. These changes could also be measured by net increases in reduced intermediate over the prestimulation baseline, taken at peak response. In order to determine how these changes in the cytochrome chain might be related to simiI IO z 2 -: + . 90 s F f
l
k + t +
/ 70
+
1
if 9 z
50 il o
001
’ 0.1
Methyl
mercuric
mg/kg
per dayx
’ I.0
chloride, 14 days
Fig. 2. The modification of pyridine nucleotide reoxidation (Phase 3) in electrically stimulated brain slices taken from animals exposed to methyl mercury. Open circle denotes average of control measurements (n = 18), filled circles are each the average of six exposed animals at each dose level except at 2 mg/kg per day where only 3/6 tissues respond in such a way as to allow measurement. Vertical bars indicate f S.E.M.
t tt I t
355
Methyl mercury and the responses of brain 0.025-
0,020.c_ E \ E
0.015-
it I 0 s R O.OlOd
+
0.005 -
t
0.L Methyl mg/kg
o!,
I I.0
mercuric chloride, per dayx14 days
Fig. 3. Initial rate of pyridine nucleotide reduction (Phase 2) with electrical stimulation and its modification by methyl mercury. Open circlesdenote average of control observations (n = 18) filled circles the average of six exposed animals at each dose level. Vertical bars indicate + S.E.M.
lar changes with the NAD(P) response, we have formed the ratio of the net increase cytochrome b absorbance over the increase in NAD(P)H absorbance at the peak of Phase 2 of the responses with the varying dosage schedules of methyl mercury. These results are presented in Figure 4. At O*Oland 0.05 mg/kg per day, the increase in NAD(P) reduction observed with electrical stimulation appeared to be paralleled by an increase’d cytochrome b response. At O-1mg/kg per day, the cytochrome b response was substantially increased relative to the NATI response. Above this dose, however, there was a progressive decline in the cytochrome h response relative to the NAD(P) response. (The 2.0 mg/kg per day dose level was not included in these data because a net reduction of NAD(P) was absent in three of the animals.) The response of cortical slices to an increase in potassium concentration (to 30 mM) in the incubation media is complex, but allows a reasonably clear visualization of the metabolic control processes involved (BULL and LUTKENHOFF, 1973). The initial response is an increase in the respiratory rate, accompanied by an oxidation of the respiratory carriers which reach peak at approximately 1 min. This phase of the response is gradually replaced
I
ok\ O.bl Methyl
mercuric
I I-O
0.1
chloride,
mg/kg
per dayx14
days
Ratio of net change in Phase 2 of the redox state of brain slice cytochrome b relative to the pyridine nucleotides with electrical stimulation (10 set train) and the effect of increasing doses of methyl mercury. Open circle denotes average of control values (n = 18), filled circles the average of six values from each exposure group. Vertical bars indicate f S.E.M. Fig. 4.
356
R. J. BULLand S. D. LUTKENHOFF
-0~0040 r -0~003QP v) : z : i d
-0.0020 -
-o~oolo-
:
Methyl mg/kg
mercuric chloride, per doyxl4 days
Fig. 5. The effect of methyl mercury on the initial oxidation of pyridine nucleotides resulting from increasing the media potassium concentration to 30 mM. Open circle denotes the average of 12 control responses, filled circles represent the average of not less than six animals at each dose level. Vertical bars indicate f S.E.M.
by a decreasing rate of oxygen consumption, a reduction of the electron carriers and an increase in the rate of aerobic glycolysis. For the purposes of this study the spectrophotometric changes were quantified by measuring net changes from the baseline at 1 and 10 min following the addition of potassium. Peak stimulation of oxygen consumption was also measured at 1 min, and the time course of the change in lactic acid production was observed over a 15-min interval. Methyl mercury, at doses above 0.15 mg/kg per day, decreased the initial oxidation of NAD(P) resulting from an increase of potassium to 30 mM (Fig. 5) although the change was statistically different from control only at the 2-O mg/kg per day dose. This effect was not reflected in any consistent changes in the resting or potassium-stimulated respiration (Table 2). Data in Figure 6 indicate that the reductive phase of the pyridine nucleotide response to elevated potassium was markedly augmented by low doses of methyl mercuric chloride. This effect of methyl mercury was maximal at 0.05 mg/kg per day and tended to decrease slightly with increasing dose. The higher level of reduced NAD(P) achieved, appeared to correspond well with the similar effect using electrical stimulation; however, the electrical responses were much more sensitive to the inhibitory effects of higher doses of methyl mercury. The effects of methyl mercury on potassium-stimulated lactic acid production are shown in Figure 7. This response was inhibited approximately 50% in rats treated with 2.0 mg/kg per day methyl mercuric chloride. Lower doses of methyl mercury appeared to have little effect on lactic acid production. DISCUSSION
Metabolism of brain tissue in vitro, has been found less active than that observed in vivo unless that tissue was in some way stimulated (MCILWAIN, 1966). Consequently, to fully Table 2. Effect of in uivo administration of methyl mercuric chloride on brain slice respiratory responses to the addition of 27 mM KC1 DOSE mg/kg per day x 14 days 0 0.01 0.05 0.5 2.0
frill
(10) 196; (8) (7)
Values presented f S.E.M.
Pre-stim. 1.7 f 2.1 * 1.6 f 1.6 + 1.4 +
0.2 03 0.3 0.3 0.3
O2 consumption pmoles/min per g Potassium stim. 1.3 & 1.3 f 1.4 f 1.3 f 1.3 *
0.1 0.3 0.2 O-4 0.2
Peak rate 3.0 3.4 3.0 2.9 27
3.51
Methyl mercury and the responses of brain
O.OlO-
0.006
-
.E E
0 -
0,006 -
I
I 0.10
Methyl mg/kg
mercuric per dayx
I.0
chloride, 14 days
Fig. 6. The modification of the potassium stimulated reduction of pyridine nucleotides in brain slices by methyl mercury. Open circle denotes the average of 12 control responses, filled circles the average of not less than six exposed animals at each dose level. Verticalbars indicate f S.E.M.
assess the implications of methyl mercury effects on cerebral energy metabolism it was necessary to examine stimulated metabolism. We chose to use two methods of stimulating the tissues, electrical pulses and elevated concentrations of potassium. While there were parallels in the effects of the two agents, the mechanisms by which they stimulate metabolism seem different on the basis of the time course of the respective responses (CUMMINS and BULL, 1971; BULL and LUTKENHOFF,1973). Additionally, metabolic responses to electrical stimulation were completely inhibited by saxitoxin or tetrodotoxin (MCILWAIN, 1967; BULL and TREVOR, 1972) whereas those following addition of potassium were not (CHAN and QUASTEL, 1967). Therefore, the use of the two different means of stimulation tested the specificity of the effects of methyl mercury. In practical terms, however, potassium stimulation allowed closer examination of the oxidative phase of the metabolic responses and electrical stimulation was better suited for examining the initial rates of intermediate reduction.
s
1.25A
F s D 0 CL
H
:: 0 boo2u 4 o.'525
P %a%, 0 30 Incubation
I 40
35 time,
I 45
J 50
min
Fig. 7. The effect of methyl mercury exposure in uiuo on potassium stimulated lactic acid production of brain slices in vitro. Potassium was added to the media bath at 30 min of incubation time and reached the collection tubes within 3 min. o Control (n = 9), 0 0.01 (n = 8), A 0.05 (n = 8), + 0.5 (a = 6), A 2.0 mg/kg per day (n = 6). S.E.M. is indicated only on the control values and experimental values which differ significantly from control.
358
R. J. BULL and
S. D.
LUTKENHOFF
Methyl mercury at very low doses (0.01-0.05 mg/kg per day) produced an enhancement of the reductive aspect of the responses to either electrical or potassium stimulation. The significance of this observation is not altogether clear but may reflect an adaptive change in brain metabolism to the presence of very small concentrations of mercury. As the dosage approached levels that produced neurological impairment, a striking inhibition of Phase 2 of the response to electrical stimulation was observed. This latter effect in terms of tissue NAD(P) responses appeared rather specific for electrical stimulation, since potassium-induced reduction of NAD(P) remained above control levels at the higher doses of methyl mercury. This was not true of potassium-stimulated lactic acid production which was appreciably inhibited at 2 mg/kg per day. Activation of aerobic glycolysis appeared to play a substantial part in the development of Phase 2 of the NAD(P) response to electrical pulses (LIPTON, 1973). On the other hand, potassium ion substantially increased the rate of pyruvate oxidation in isolated brain mitochondria (NICKLAS,CLARKand WILLIAMSON,1971), in addition to its well known effects on glycolysis (BYGRAVE,1967). It is tempting to speculate that an increased activation of mitochondrial oxidations was responsible for enhancement of the reductive aspects of both methods of stimulation and that a decrease “responsiveness” of the glycolytic pathway to activation was responsible for the inhibition of Phase 2 of the responses to electrical stimulation and potassium-stimulated lactic acid production. In this regard, it should be pointed out that the failure of methyl mercury at the higher doses to inhibit potassium-induced reduction of NAD(P) may be related to the observed inhibition of NAD(P)H oxidation using electrical stimulation rather than a continued enhancement of substrate oxidation. This possibility would be consistent with the inhibition of potassium-stimulated lactic acid production at the highest dose of methyl mercury. YOSHINOet al. (1966) have shown that inhibition of potassium-stimulated lactic acid production under aerobic conditions in cortex slices taken from animals exposed to a large dose of methyl mercury was not accompanied by an inhibition of the anaerobic glycolytic rate. This implied that methyl mercury affects the control of glycolysis and need not alter the absolute activities of the individual enzymes of the pathway. Substantial changes in the levels of the glycolytic intermediates have been observed in the brains of rats and mice exposed to low doses of methyl mercury (PATTERSONand USHER, 1971; SALVATERRA, LOWN, MORGANTIand MASSARO,1973). Alterations in the kinetics of NAD(P)H oxidation were observed with both electrical and potassium stimulation. Phase 3 of the response to electrical stimulation was significantly prolonged at doses of 0.05 mg/kg per day. At intermediate doses (0.25 and 05 mg/kg per day), this change was associated with reduced responses in the cytochrome chain indicating some impairment of reducing equivalent flow between NAD(P) and cytochrome 6. Significant inhibitionofthe initialNAD(P)H oxidation produced by increasing the media potassium concentration to 30 mM was observed only at the highest dose, 2 mg/kg per day, and was not accompanied by a significant change in potassium-stimulated respiration. This latter observation was in contrast to the results of YOSHINOet al. (1966) who observed a significant depression of potassium-stimulated respiration in brain slices taken from animals treated with methyl mercury. Two factors probably account for this discrepancy. First the dose of methyl mercury used by these workers was 75 mg/kg as the thioacetamide salt amounting to 2.2 times the maximum total dose administered in the present study when calculated as mercury. Secondly, 30 mM potassium does not maximally stimulate cortical slice respiration (BULL and CUMMINS,1973). YOSHINOet al. (1966) employed a concentration of 50 mM potassium. Under our conditions, potassium at 30 mM produced high rates of respiration but only for a limited period of time, implying that sufficient reserve of reducing equivalent was present to fuel the short burst of respiration observed. However, methyl mercury inhibition of the initial NAD(P)H oxidation produced by potassium would probably result in a decrease in the larger respiratory response observed with higher concentrations of potassium. The prolongation of Phase 3 of the responses to electrical stimulation was not accompanied by changes in oxygen consumption of the tissues. This suggests that either oxidation ofa particular pool of NAD(P)H was inhibited, or that the kinetics of a different pool predominates in the metabolic responses to stimulation of brain slices taken from animals
Methyl mercury and the responses of brain
359
exposed to methyl mercury. These observations may be related to the marked changes indicated in the a-glycerophosphate shuttle in the brains of rats exposed to small amounts of methyl mercury (PATERSON and USHER, 1971). From our results the conclusion may be reached that alterations in intermediary metabolism of brain occur at doses of methyl mercury far below those producing overt toxicity in rats. Only at the highest dose, 2 mg/kg per day for 14 days did significant losses in weight occur. At this dose too, the first changes in neurological function were observed. Cortex levels of mercury observed at this dosage were also consistent with previous approximations of brain levels of mercury which produce overt neurological deficits (FRIBERG, 1971). The changes produced by methyl mercury in cortical slices taken from exposed animals involved responses to changes in “functional” state imposed by either electrical or ionic stimulation. This type of manipulation was used to raise the metabolic activity of the normally electrically quiescent cortical slices to those observed in uiuo. The alterations produced by methyl mercury in this “active” state must be considered when attempting to elucidate the mechanisms of methyl mercury induced damage to the central nervous system. REFERENCES BULL,R. J. and CUMMINS,J. T. (1973). Influence of potassium on the steady-state redox potential of the electron transport chain in slices of rat cerebral cortex and the effect of ouabain. J. Neurochem. 21: 923-937. BULL, R. J. and LIJTKENHOFF, S. D. (1973). Early changes in respiration aerobic glycolysis and cellular NAD(P)H in slices of rat cerebral cortex exposed to elevated concentrations of potassium. J. Neurochem. 21: 913-922. BULL,R. J. and TREVOR,A. J. (1972). Saxitoxin, Tetrodotoxin and the metabolism and cation flux in isolated cerebral tissues. J. Neurochem. 19: 999-1009. BYGRAVE,F. L. (1967). The ionic environment and metabolic control. Nature, Lond. 214: 667-671. CHAN, S. L. and QUASTEL,J. H. (1967). Tetrodotoxin: Effects on brain metabolism in vitro. Science, N.Y. 156: 1752-1753. CHANCE,B. and HOLLUNGER, G. (1963). Inhibition of electron and energy transfer in mitochondria. I. Effects of amytal, thiopental, rotenone, progesterone, and methylene glycol. J. hiol. Chem. 278: 418-431. CLARKSON,T. W. (1972). Recent advances in the toxicology of mercury with emphasis on the alkylmercurials. Crit Reo Toxicol. 1: 203-234. CUMMINS,J. T. and BULL,R. J. (1971). Spectrophotometric measurements of metabolic responses in isolated rat brain cortex. Biochim. biophys. Acta 253: 29-38. E’RIBERG, L., Chairman (1971). Methyl Mercury in Fish. A Toxicologic-Epidemiologic Evaluation ofRisks. Nordisk Hygienisk Tidskrift, Supplement 4-National Institute of Public Health, Stockholm, Sweden. HOHORST,H. J. (1963). Lactate dehydrogenase. In: Methods of Enzymatic Analysis (BERGMEYER, H. U., Ed.), p. 266. Academic Press, New York. JENSEN,S. and JERNELOV, A. (1969). Biological methylation of mercury in aquatic organisms. Nature, Lond. 223: 753-754.
KLEIN,R., HERMAN,S. P., BRUBAKER, P. E. and LUCIER,G. W. (1972). A model of acute methyl mercury intoxication in rats. Archs Path., 93: 408418. LIPTON,P. (1973). Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices. Biochem. J. 136: 999-1009.
MCILWAIN,H. (1967). Tetrodotoxin and the cation content, excitability and metabolism of isolated mammalian cerebral tissues. Biochem. Pharmac. 16: 13891396. MCILWAIN, H. (1966). Biochemistry and the Central Nervous System, p. 61. Little, Brown & Co., Boston. NICKLAS,W. J., CLARK,J. B. and WILLIAMSON, J. R. (1971). Metabolism of rat brain mitochondria. Studies on the potassium ion-stimulated oxidation of pyruvate. Biochem. J. 123: 83-95. PATERSON,R. A. and USHER,D. R. (1971). Acute toxicity of methyl mercury on glycolytic intermediates and adenine nucleotides of rat brain. Life Sci. 10: 121-128. RIVERS,J. B., PEARSON,J. E. and SCHULTZ,C. D. (1972). Total and organic mercury in marine fish. BuK Environ. Contam. Toxicol. 8: 257-266.
SALVA~RRA,P., LOWN, B., MORGANTI,J. and MASSARO,E. J. (1973). Alterations in neurochemical and behavioural parameters in the mouse induced by low doses of methyl mercury. Acta pharmac. tox. 33: 177-190. SHALIT,M. N., BELLER,A. J., and FEINSOD,M. (1972). Clinical equivalents of cerebral oxygen consumption in coma. Neurology, Minneap. 22: 155160. WESTOO,G. (1969). Methylmercury compounds in animal foods. In: Chemical Fallout, Current Research on Persistent Pesticides (MILLER,M. W. and BERG,C. G., Eds.), pp. 75-93. C. C. Thomas, Springfield. YOSHINO, Y., MOZAI,T. and NAKAO,K. (1966). Biochemical changes in the brain in rats poisoned with an alkymercury compound, with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurothem. 12: 122>1230.
1975, 14. 351-359. Pergamon
Press.
Printed
in Gt. Britain.
CHANGES IN THE METABOLIC RESPONSES OF BRAIN TISSUE TO STIMULATION, IN VITRO, PRODUCED BY IN VII/O ADMINISTRATION OF METHYL MERCURY R. J. BULL and S. D. LUTKENHOFF Water Supply Research Laboratory, National Environmental Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 (Accepted
11September 1974)
Summary~Electrical stimulation of rat cerebral cortex slices produced responses in the redox state of nicotinamide-adenine dinucleotides (NAD(P))* and cytochrome intermediates which were measured spectrophotometrically. These responses could be divided into three distinct phases: (I) an initial oxidative phase, followed by (2) a reductive phase and (3) a relaxation to the prestimulation baseline. Adult SpragueeDawley rats were exposed to intraperitoneal injections of methyl mercuric chloride in doses ranging from 0.005 to 2.0 mg/kg per day for a period of 14 days. Methyl mercury enhanced the reductive response at low doses and inhibited at higher doses. At doses of 0.05 mg/kg per day and higher, methyl mercury significantly inhibited the reoxidation of NAD(P) reduced by electrical stimulation. An increase in the media potassium concentration from 3 to 30 mM resulted in an initial oxidation followed by a net reduction of NAD(P). Inhibition of the initial oxidative phase of the response to potassium stimulation became evident at doses above 0.15 mg/kg per day. The reductive response of the NAD(P) to addition of potassium was also enhanced by low doses of methyl mercury. In contrast to results with electrical stimulation, inhibition of this response at high doses was not as apparent. However, aerobic glycolytic rate in response to addition of potassium was found to be inhibited at 2.0 mg/kg per day. These results are discussed in terms of the necessary integration of energy metabolism with the function of brain.
The occurrence of methyl mercury in the aquatic environment has been extensively documented in recent years (see, e.g. JENSEN and JERNELOV, 1969). Mercury in this form has been shown to gain access to the food chain of man (WESTOO,1969, RIVERS, PEARSON and SHULTZ, 1972). These findings have stimulated interest in the toxicological properties peculiar to alkylated derivatives of mercury. Although it is generally agreed that the nervous system is predominately involved in the toxicity of the alkyl mercurials, there is little information dealing with methyl mercury-induced changes in the biochemistry and physiology of the central nervous system that are related to a gradation of exposures over a time period. Data derived from various investigations (see, e.g. CLARKSON, 1972) have documented important differences in the physiological availability and distribution of these compounds relative to the inorganic salts of mercury. As a consequence, considerable emphasis has been placed on these differences to account for the clearly different toxic symptoms associated with the alkyl mercurials. Although one can show a relative increase of mercury in the central nervous system when methyl mercury, as opposed to the inorganic mercurials, is administered to animals, the brain still remains low in mercury concentration relative to liver and kidney with either acute or chronic exposures (FRIBERG, 1971). While this does not negate the importance of the distributional differences as being partially responsible for the predominance of nervous system symptoms with the alkyl mercurials, it is apparent that the effects are also a result of a metabolism peculiar to nerve tissue. One way in which brain metabolism has long been recognized as unique, is that adequate amounts of glucose and oxygen must be supplied continuously to avoid compromise of
* The abbreviation NAD(P) is used to designate both forms of the nicotinamide-adenine dinucleotides (i.e. NAD and NADP) because spectral differentiation of the compounds is not possible. In brain, the changes observed are most likely ascribed to NAD for a variety of reasons, but this cannot be regarded as proven. 351
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R. J. BULL andS. D. LUTKENHOFF
function (SHALIT,BELLERand FEINSOD,1972). Previous reports have indicated that methyl mercury produces alterations in glucose metabolism in the brain of rats (PATERSONand USHER, 197 1; YOSHINO,MOZAI and NAKAO, 1966). On the basis of these earlier reports, we decided to examine the relationship between the functional activity and energy metabolism in slices of cerebral cortex taken from rats exposed to methyl mercury. Our approach depends on the kinetics of changes induced in various metabolic parameters in the transition from a resting to an active state (depolarization). Methyl mercury has been found to produce rather complex changes in these metabolic responses suggesting that the coordination of energy metabolism to functional activity (i.e. metabolic control) has been impaired. METHODS
Treatment of’animals Adult Sprague-Dawley rats (450-600 g) were injected intraperitoneally with methyl mercuric chloride, dissolved in distilled water, at doses ranging from 0.005 to 2-Omg/kg per day as the salt (20% less as Hg) for a 14-day period. Control animals were injected with an equal volume of distilled water. All solutions were adjusted to a pH of 5.5. Food and water were supplied ad lib. On the fifteenth day the animals were sacrificed. Preparation and incubation o~tissues Brain slices were prepared from the surface of each hemisphere of the cerebral cortex using a Stadie-Riggs microtome blade and glass guide (BULL and LUTKENHOFF,1973). Slices used for measurement of oxygen uptake and aerobic glycolytic rate were touched to a clean glass surface to remove adhering media and were weighed to the nearest 05 mg. Determination of the cross-sectional area and weight of eight brain slices was used to calculate their average thickness, assuming equal specific gravity with water. The figure obtained was 0.41 3_ 0.02 mm (S.E.M.). In all cases, the incubation media initially consisted of (MM); NaCl, 127; Mg&, 1.3; NaH,PO1, l-3; CaCl,, 0.75; NaHCOs, 26; and KCl, 3.0. Where indicated, 27 mM KC1 was added to bring the media potassium concentration to 30 mM. The incubation media was continuously oxygenated with 95% oxygen plus 5% carbon dioxide, thus maintaining pH at 7~4.Temperature of the incubation media was maintained at 37.0 f 0.2”C in both the spectrophotometric cuvette and the respirometer. The time course of each experiment consisted of a 30-min acclimatization period prior to the beginning of stimulation. At that point either the sequence of measurements of the responses to electrical stimulation was begun or potassium was added to stimulate the tissues. Experiments using electrical stimuIation lasted about one additional hour, whereas those utilizing potassium stimulation lasted for only another 20 min. In both cases, this was well within the survival time of the tissues, since preliminary experiments had indicated that the responses to electrical stimulation were mainlined without decrement for more than 2 hr. Absorbance of the ix-band of the cytochromes is also well-maintained over a similar time interval (BULL and CUMMINS,1973). Measurement of metabolic responses Spectrophotometric measurement of the responses of the respiratory intermediates of brain slices has been previously reported (CUMMINSand BULL, 1971; BULL and LUTKENHOFF,1973) and is based upon the pioneering work of Chance and associates in this area over the last 20 yr (see CHANCEand HOLLUNGER,1963). The responses to electrical stimulation were measured in each slice and in the following sequence 605-630 nm (cytochrome a), 562-575 nm (cytochrome b), 550-540 nm (cytochromes c + c,), 465-510 nm (flavoproteins), 445-455 nm (cytochrome a3) and 340-374 nm (nicotinamide-adenine dinucleotides, NAD(P)H). Electric field stimulation, consisting of biphasic square wave pulses of 7 V amplitude, 60 Hz, 0.5 msec pulse duration, was applied through enamelled silver wire electrodes. A Grass S88 stimulator, equipped with two stimulus isolation units, was used to generate the pulses. Pulse characteristics were continuously monitored on a cath-
Methyl mercury and the responses of brain
353
ode ray tube. The standard stimulus was a train of 10-Oset duration. To answer the question of how reproducible the responses to electrical stimulation might be, three separate control groups of six animals each were utilized. No significant differences were noted between the three groups of animals. Consequently, results were combined and are treated here as a single group. A similar operation was used with the potassium stimulated responses, but only two groups of six animals each were utilized. Each experimental group had six animals for studying the effects of electrically stimulated responses. In the case of potassium stimulation, the numbers utilized in each group involved a minimum of six animals per group for each parameter examined (i.e. not all parameters were looked at with each exposed animal). Cortex slices from methyl mercury exposed animals were also stimulated by increasing the media potassium concentration to 30 mM. The spectrophotometric, respiratory, and glycolytic responses to such stimulation have been previously described (BULL and LUTKENHOFF, 1973). The respiratory response of brain slices to addition of potassium was measured polarographically in a perfusion system in parallel experiments. Glycolytic rate of the tissues was followed by sampling the effluent of this latter apparatus at 2-min intervals and enzymatically assaying for lactic acid according to the method of HOHORST (1963). Mercury
determination
The mercury gradient observed in the cerebral cortex of rats exposed under the above conditions was determined in a separate experiment. Three groups of five rats were injected with 0.02, 0.2 and 2 mg/kg per day for 14 days. A fourth group was injected with distilled water over the same time period and served as control. Upon sacrifice (on 15th day) the brain was removed, subcortical matter dissected away, and cleaned of blood vessels with a cotton swab moistened with incubation media. Each hemisphere of the cerebral cortex (400-500mg) served as a sample. The tissues were digested in concentrated sulphuric (30 ml) and nitric acids (5 ml) and 2% potassium persulphate (10 ml) for 16 hr at 60°C. Mercury content was measured using flameless atomic absorption spectrophotometry. Average recovery was found to be 103%. RESULTS
As previously reported by other investigators (see, e.g. KLEIN, HERMAN, BRUBAKER and LUCIER, 1972), methyl mercury in sufficiently high doses, produced substantial losses in body weight. Consequently, animals on the 14&y intraperitoneal schedule of methyl mercury were weighed daily to get some measure of the gross toxicity of each dosage regimen. At 05 mg/kg per day, a significant decrease in the weight gained over this period was observed and at 2.0 mg/kg per day substantial losses in weight were recorded. Doses lower than 0.5 mg/kg per day had no observable effect on body weight. Table 1 indicates the concentrations of mercury reached in the cerebral cortex of animals dosed with methyl mercuric chloride in the range used in the present study. Mercury content of control animals was found to be quite low, barely greater than reagent blank values. Significant elevations in the level of mercury were found at 0.02 mg/kg per day. Higher doses progressively increased the mercury content of the cerebral cortex. Figure 1 illustrates the basic features of a response of the respiratory intermediates of cortical slices to electrical stimulation. The response pictured was recorded at 34&374 nm corresponding to NAD(P)H.Thisresponseand those measured at the other wavelength pairs Table 1. Accumulation of mercury in the cerebral cortex of rats given intraperitoneal injections of methyl mercuric chloride Dose of methyl mercuric chloride mg/kg per day x 14 0 0.02 0.2 2.0
Concentration of mercury in cerebral cortex (pg/g) 0.0049 0.0258 0.778 9.88
f + f f
Values represent the mean of five animals f S.E.M.
0+)020 O%I44 0.057 0.87
354
R.J. BULL and S.D. LUTKENHOFF
II I Phase
I
1
1
I2 1
I 3
1
Ttme
1
1
1
I
(min)
Fig. 1. Normal response of brain slices to a IO-set period of electrical stimulation illustrating its three phases. Measuring wavelengths, 340-374 nm.
can be divided into three phases for purposes of quantification; an initial oxidative phase, followed by a substantial reductive phase and a third phase of relaxation of the response back to the prestimulation baseline. For the purposes of this paper, data will be presented on the second and third phases. The initial oxidation was too rapid and variable for accurate characterization with the available instrumentation. The remainder of the response can be adequately described by two measurements. (1) An initial rate of reduction, which was measured as the slope of the second phase relative to baseline, and (2) a relaxation time of the response, describing the reoxidation of intermediate to prestimulation levels. Reoxidation of the NAD(P) reduced by electrical stimulation (Phase 3) was significantly slowed with doses of methyl mercuric chloride as low as 0.05 mg/kg per day (Fig. 2). This effect appeared to be maximal at 0.5 mg/kg per day, but was complicated at 2.0 mg/kg per day by a loss of the normal net reduction of NAD(P) in response to electrical stimulation in three of six animals. Instead, the responses were purely oxidative in direction relative to the prestimulation baseline, and allowed no measurement of the time constant to be made. The value indicated in Figure 2 for the 2.0 mg/kg per day dose was the average of the remaining three tissues. This effect appeared specific for the NAD(P) response. Only at 2 mg/kg per day was any alteration observed in the reoxidation of the cytochrome intermediates. Even these results were inconsistent and only in the case of cytochrome a (605630 nm) was the change statistically significant (i.e. increased from 47 to 85 set; P < 0.05). Lower doses of methyl mercuric chloride (0.01 mg/kg per day) were seen to substantially increase the rate of NAD(P) reduction with electrical stimulation (Fig. 3), but higher doses ( > 0.25 mg/kg per day) progressively decreased the rate of NAD(P) reduction (Phase 2) seen with electrical stimulation. Similar trends were observed in the cytochrome chain, i.e. a tendency for a more rapid reduction of intermediate at low doses followed by an apparent inhibition of this phase at higher doses. These changes could also be measured by net increases in reduced intermediate over the prestimulation baseline, taken at peak response. In order to determine how these changes in the cytochrome chain might be related to simiI IO z 2 -: + . 90 s F f
l
k + t +
/ 70
+
1
if 9 z
50 il o
001
’ 0.1
Methyl
mercuric
mg/kg
per dayx
’ I.0
chloride, 14 days
Fig. 2. The modification of pyridine nucleotide reoxidation (Phase 3) in electrically stimulated brain slices taken from animals exposed to methyl mercury. Open circle denotes average of control measurements (n = 18), filled circles are each the average of six exposed animals at each dose level except at 2 mg/kg per day where only 3/6 tissues respond in such a way as to allow measurement. Vertical bars indicate f S.E.M.
t tt I t
355
Methyl mercury and the responses of brain 0.025-
0,020.c_ E \ E
0.015-
it I 0 s R O.OlOd
+
0.005 -
t
0.L Methyl mg/kg
o!,
I I.0
mercuric chloride, per dayx14 days
Fig. 3. Initial rate of pyridine nucleotide reduction (Phase 2) with electrical stimulation and its modification by methyl mercury. Open circlesdenote average of control observations (n = 18) filled circles the average of six exposed animals at each dose level. Vertical bars indicate + S.E.M.
lar changes with the NAD(P) response, we have formed the ratio of the net increase cytochrome b absorbance over the increase in NAD(P)H absorbance at the peak of Phase 2 of the responses with the varying dosage schedules of methyl mercury. These results are presented in Figure 4. At O*Oland 0.05 mg/kg per day, the increase in NAD(P) reduction observed with electrical stimulation appeared to be paralleled by an increase’d cytochrome b response. At O-1mg/kg per day, the cytochrome b response was substantially increased relative to the NATI response. Above this dose, however, there was a progressive decline in the cytochrome h response relative to the NAD(P) response. (The 2.0 mg/kg per day dose level was not included in these data because a net reduction of NAD(P) was absent in three of the animals.) The response of cortical slices to an increase in potassium concentration (to 30 mM) in the incubation media is complex, but allows a reasonably clear visualization of the metabolic control processes involved (BULL and LUTKENHOFF, 1973). The initial response is an increase in the respiratory rate, accompanied by an oxidation of the respiratory carriers which reach peak at approximately 1 min. This phase of the response is gradually replaced
I
ok\ O.bl Methyl
mercuric
I I-O
0.1
chloride,
mg/kg
per dayx14
days
Ratio of net change in Phase 2 of the redox state of brain slice cytochrome b relative to the pyridine nucleotides with electrical stimulation (10 set train) and the effect of increasing doses of methyl mercury. Open circle denotes average of control values (n = 18), filled circles the average of six values from each exposure group. Vertical bars indicate f S.E.M. Fig. 4.
356
R. J. BULLand S. D. LUTKENHOFF
-0~0040 r -0~003QP v) : z : i d
-0.0020 -
-o~oolo-
:
Methyl mg/kg
mercuric chloride, per doyxl4 days
Fig. 5. The effect of methyl mercury on the initial oxidation of pyridine nucleotides resulting from increasing the media potassium concentration to 30 mM. Open circle denotes the average of 12 control responses, filled circles represent the average of not less than six animals at each dose level. Vertical bars indicate f S.E.M.
by a decreasing rate of oxygen consumption, a reduction of the electron carriers and an increase in the rate of aerobic glycolysis. For the purposes of this study the spectrophotometric changes were quantified by measuring net changes from the baseline at 1 and 10 min following the addition of potassium. Peak stimulation of oxygen consumption was also measured at 1 min, and the time course of the change in lactic acid production was observed over a 15-min interval. Methyl mercury, at doses above 0.15 mg/kg per day, decreased the initial oxidation of NAD(P) resulting from an increase of potassium to 30 mM (Fig. 5) although the change was statistically different from control only at the 2-O mg/kg per day dose. This effect was not reflected in any consistent changes in the resting or potassium-stimulated respiration (Table 2). Data in Figure 6 indicate that the reductive phase of the pyridine nucleotide response to elevated potassium was markedly augmented by low doses of methyl mercuric chloride. This effect of methyl mercury was maximal at 0.05 mg/kg per day and tended to decrease slightly with increasing dose. The higher level of reduced NAD(P) achieved, appeared to correspond well with the similar effect using electrical stimulation; however, the electrical responses were much more sensitive to the inhibitory effects of higher doses of methyl mercury. The effects of methyl mercury on potassium-stimulated lactic acid production are shown in Figure 7. This response was inhibited approximately 50% in rats treated with 2.0 mg/kg per day methyl mercuric chloride. Lower doses of methyl mercury appeared to have little effect on lactic acid production. DISCUSSION
Metabolism of brain tissue in vitro, has been found less active than that observed in vivo unless that tissue was in some way stimulated (MCILWAIN, 1966). Consequently, to fully Table 2. Effect of in uivo administration of methyl mercuric chloride on brain slice respiratory responses to the addition of 27 mM KC1 DOSE mg/kg per day x 14 days 0 0.01 0.05 0.5 2.0
frill
(10) 196; (8) (7)
Values presented f S.E.M.
Pre-stim. 1.7 f 2.1 * 1.6 f 1.6 + 1.4 +
0.2 03 0.3 0.3 0.3
O2 consumption pmoles/min per g Potassium stim. 1.3 & 1.3 f 1.4 f 1.3 f 1.3 *
0.1 0.3 0.2 O-4 0.2
Peak rate 3.0 3.4 3.0 2.9 27
3.51
Methyl mercury and the responses of brain
O.OlO-
0.006
-
.E E
0 -
0,006 -
I
I 0.10
Methyl mg/kg
mercuric per dayx
I.0
chloride, 14 days
Fig. 6. The modification of the potassium stimulated reduction of pyridine nucleotides in brain slices by methyl mercury. Open circle denotes the average of 12 control responses, filled circles the average of not less than six exposed animals at each dose level. Verticalbars indicate f S.E.M.
assess the implications of methyl mercury effects on cerebral energy metabolism it was necessary to examine stimulated metabolism. We chose to use two methods of stimulating the tissues, electrical pulses and elevated concentrations of potassium. While there were parallels in the effects of the two agents, the mechanisms by which they stimulate metabolism seem different on the basis of the time course of the respective responses (CUMMINS and BULL, 1971; BULL and LUTKENHOFF,1973). Additionally, metabolic responses to electrical stimulation were completely inhibited by saxitoxin or tetrodotoxin (MCILWAIN, 1967; BULL and TREVOR, 1972) whereas those following addition of potassium were not (CHAN and QUASTEL, 1967). Therefore, the use of the two different means of stimulation tested the specificity of the effects of methyl mercury. In practical terms, however, potassium stimulation allowed closer examination of the oxidative phase of the metabolic responses and electrical stimulation was better suited for examining the initial rates of intermediate reduction.
s
1.25A
F s D 0 CL
H
:: 0 boo2u 4 o.'525
P %a%, 0 30 Incubation
I 40
35 time,
I 45
J 50
min
Fig. 7. The effect of methyl mercury exposure in uiuo on potassium stimulated lactic acid production of brain slices in vitro. Potassium was added to the media bath at 30 min of incubation time and reached the collection tubes within 3 min. o Control (n = 9), 0 0.01 (n = 8), A 0.05 (n = 8), + 0.5 (a = 6), A 2.0 mg/kg per day (n = 6). S.E.M. is indicated only on the control values and experimental values which differ significantly from control.
358
R. J. BULL and
S. D.
LUTKENHOFF
Methyl mercury at very low doses (0.01-0.05 mg/kg per day) produced an enhancement of the reductive aspect of the responses to either electrical or potassium stimulation. The significance of this observation is not altogether clear but may reflect an adaptive change in brain metabolism to the presence of very small concentrations of mercury. As the dosage approached levels that produced neurological impairment, a striking inhibition of Phase 2 of the response to electrical stimulation was observed. This latter effect in terms of tissue NAD(P) responses appeared rather specific for electrical stimulation, since potassium-induced reduction of NAD(P) remained above control levels at the higher doses of methyl mercury. This was not true of potassium-stimulated lactic acid production which was appreciably inhibited at 2 mg/kg per day. Activation of aerobic glycolysis appeared to play a substantial part in the development of Phase 2 of the NAD(P) response to electrical pulses (LIPTON, 1973). On the other hand, potassium ion substantially increased the rate of pyruvate oxidation in isolated brain mitochondria (NICKLAS,CLARKand WILLIAMSON,1971), in addition to its well known effects on glycolysis (BYGRAVE,1967). It is tempting to speculate that an increased activation of mitochondrial oxidations was responsible for enhancement of the reductive aspects of both methods of stimulation and that a decrease “responsiveness” of the glycolytic pathway to activation was responsible for the inhibition of Phase 2 of the responses to electrical stimulation and potassium-stimulated lactic acid production. In this regard, it should be pointed out that the failure of methyl mercury at the higher doses to inhibit potassium-induced reduction of NAD(P) may be related to the observed inhibition of NAD(P)H oxidation using electrical stimulation rather than a continued enhancement of substrate oxidation. This possibility would be consistent with the inhibition of potassium-stimulated lactic acid production at the highest dose of methyl mercury. YOSHINOet al. (1966) have shown that inhibition of potassium-stimulated lactic acid production under aerobic conditions in cortex slices taken from animals exposed to a large dose of methyl mercury was not accompanied by an inhibition of the anaerobic glycolytic rate. This implied that methyl mercury affects the control of glycolysis and need not alter the absolute activities of the individual enzymes of the pathway. Substantial changes in the levels of the glycolytic intermediates have been observed in the brains of rats and mice exposed to low doses of methyl mercury (PATTERSONand USHER, 1971; SALVATERRA, LOWN, MORGANTIand MASSARO,1973). Alterations in the kinetics of NAD(P)H oxidation were observed with both electrical and potassium stimulation. Phase 3 of the response to electrical stimulation was significantly prolonged at doses of 0.05 mg/kg per day. At intermediate doses (0.25 and 05 mg/kg per day), this change was associated with reduced responses in the cytochrome chain indicating some impairment of reducing equivalent flow between NAD(P) and cytochrome 6. Significant inhibitionofthe initialNAD(P)H oxidation produced by increasing the media potassium concentration to 30 mM was observed only at the highest dose, 2 mg/kg per day, and was not accompanied by a significant change in potassium-stimulated respiration. This latter observation was in contrast to the results of YOSHINOet al. (1966) who observed a significant depression of potassium-stimulated respiration in brain slices taken from animals treated with methyl mercury. Two factors probably account for this discrepancy. First the dose of methyl mercury used by these workers was 75 mg/kg as the thioacetamide salt amounting to 2.2 times the maximum total dose administered in the present study when calculated as mercury. Secondly, 30 mM potassium does not maximally stimulate cortical slice respiration (BULL and CUMMINS,1973). YOSHINOet al. (1966) employed a concentration of 50 mM potassium. Under our conditions, potassium at 30 mM produced high rates of respiration but only for a limited period of time, implying that sufficient reserve of reducing equivalent was present to fuel the short burst of respiration observed. However, methyl mercury inhibition of the initial NAD(P)H oxidation produced by potassium would probably result in a decrease in the larger respiratory response observed with higher concentrations of potassium. The prolongation of Phase 3 of the responses to electrical stimulation was not accompanied by changes in oxygen consumption of the tissues. This suggests that either oxidation ofa particular pool of NAD(P)H was inhibited, or that the kinetics of a different pool predominates in the metabolic responses to stimulation of brain slices taken from animals
Methyl mercury and the responses of brain
359
exposed to methyl mercury. These observations may be related to the marked changes indicated in the a-glycerophosphate shuttle in the brains of rats exposed to small amounts of methyl mercury (PATERSON and USHER, 1971). From our results the conclusion may be reached that alterations in intermediary metabolism of brain occur at doses of methyl mercury far below those producing overt toxicity in rats. Only at the highest dose, 2 mg/kg per day for 14 days did significant losses in weight occur. At this dose too, the first changes in neurological function were observed. Cortex levels of mercury observed at this dosage were also consistent with previous approximations of brain levels of mercury which produce overt neurological deficits (FRIBERG, 1971). The changes produced by methyl mercury in cortical slices taken from exposed animals involved responses to changes in “functional” state imposed by either electrical or ionic stimulation. This type of manipulation was used to raise the metabolic activity of the normally electrically quiescent cortical slices to those observed in uiuo. The alterations produced by methyl mercury in this “active” state must be considered when attempting to elucidate the mechanisms of methyl mercury induced damage to the central nervous system. REFERENCES BULL,R. J. and CUMMINS,J. T. (1973). Influence of potassium on the steady-state redox potential of the electron transport chain in slices of rat cerebral cortex and the effect of ouabain. J. Neurochem. 21: 923-937. BULL, R. J. and LIJTKENHOFF, S. D. (1973). Early changes in respiration aerobic glycolysis and cellular NAD(P)H in slices of rat cerebral cortex exposed to elevated concentrations of potassium. J. Neurochem. 21: 913-922. BULL,R. J. and TREVOR,A. J. (1972). Saxitoxin, Tetrodotoxin and the metabolism and cation flux in isolated cerebral tissues. J. Neurochem. 19: 999-1009. BYGRAVE,F. L. (1967). The ionic environment and metabolic control. Nature, Lond. 214: 667-671. CHAN, S. L. and QUASTEL,J. H. (1967). Tetrodotoxin: Effects on brain metabolism in vitro. Science, N.Y. 156: 1752-1753. CHANCE,B. and HOLLUNGER, G. (1963). Inhibition of electron and energy transfer in mitochondria. I. Effects of amytal, thiopental, rotenone, progesterone, and methylene glycol. J. hiol. Chem. 278: 418-431. CLARKSON,T. W. (1972). Recent advances in the toxicology of mercury with emphasis on the alkylmercurials. Crit Reo Toxicol. 1: 203-234. CUMMINS,J. T. and BULL,R. J. (1971). Spectrophotometric measurements of metabolic responses in isolated rat brain cortex. Biochim. biophys. Acta 253: 29-38. E’RIBERG, L., Chairman (1971). Methyl Mercury in Fish. A Toxicologic-Epidemiologic Evaluation ofRisks. Nordisk Hygienisk Tidskrift, Supplement 4-National Institute of Public Health, Stockholm, Sweden. HOHORST,H. J. (1963). Lactate dehydrogenase. In: Methods of Enzymatic Analysis (BERGMEYER, H. U., Ed.), p. 266. Academic Press, New York. JENSEN,S. and JERNELOV, A. (1969). Biological methylation of mercury in aquatic organisms. Nature, Lond. 223: 753-754.
KLEIN,R., HERMAN,S. P., BRUBAKER, P. E. and LUCIER,G. W. (1972). A model of acute methyl mercury intoxication in rats. Archs Path., 93: 408418. LIPTON,P. (1973). Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices. Biochem. J. 136: 999-1009.
MCILWAIN,H. (1967). Tetrodotoxin and the cation content, excitability and metabolism of isolated mammalian cerebral tissues. Biochem. Pharmac. 16: 13891396. MCILWAIN, H. (1966). Biochemistry and the Central Nervous System, p. 61. Little, Brown & Co., Boston. NICKLAS,W. J., CLARK,J. B. and WILLIAMSON, J. R. (1971). Metabolism of rat brain mitochondria. Studies on the potassium ion-stimulated oxidation of pyruvate. Biochem. J. 123: 83-95. PATERSON,R. A. and USHER,D. R. (1971). Acute toxicity of methyl mercury on glycolytic intermediates and adenine nucleotides of rat brain. Life Sci. 10: 121-128. RIVERS,J. B., PEARSON,J. E. and SCHULTZ,C. D. (1972). Total and organic mercury in marine fish. BuK Environ. Contam. Toxicol. 8: 257-266.
SALVA~RRA,P., LOWN, B., MORGANTI,J. and MASSARO,E. J. (1973). Alterations in neurochemical and behavioural parameters in the mouse induced by low doses of methyl mercury. Acta pharmac. tox. 33: 177-190. SHALIT,M. N., BELLER,A. J., and FEINSOD,M. (1972). Clinical equivalents of cerebral oxygen consumption in coma. Neurology, Minneap. 22: 155160. WESTOO,G. (1969). Methylmercury compounds in animal foods. In: Chemical Fallout, Current Research on Persistent Pesticides (MILLER,M. W. and BERG,C. G., Eds.), pp. 75-93. C. C. Thomas, Springfield. YOSHINO, Y., MOZAI,T. and NAKAO,K. (1966). Biochemical changes in the brain in rats poisoned with an alkymercury compound, with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurothem. 12: 122>1230.