- Email: [email protected]
The effect of chronic exposure to sodium nitrite on the electroencephalogram of rats
The
Effect
of Chronic
Exposure
to Sodium
on the Electroencephalogram K. BXHROOZI, Encironmstul School-Hebrew
S. ROBINSON,
Nitrite
of Rats
N. GHUENER,
AND H. I. SHUVAL
Health
L,ahoratory, Department of Medical Ecology, Hndassah Medical University, Jerusalem, Israel and Talbieh Psychiatric Hospital. Jerusa!em, Israel Received
February
2, 1972
Brain electrical changes were observed in rats chronically treated with NaNOl in drinking water ranging in concentration from 100 to 2000 mg/l. The brain electrical patterns did not return to normal after the withdrawal of NaNO?, even at the lowest exposure dose of 100 mg/liter which was equivalent to a daily dose of 14 mg/kg. Each concentration of NaN02 drinking solution presented a characteristic pictnrcb of its own, i.e., in the nnfolding electroencephalogram several types of changes brcame discernible tallying with the specific dosages of NaKO> drinking solutions consumed by the rats, having a strong common denominator. These brain electrical activity changes are discussed in this paper. The ever-increasing concentration of nitrogenous compounds in water, food, and air has become a great public health concern all over the world. Organic solids and liquid wastes and nitrogenous chemicals and fertilizers used extensively
in agriculture contribute greatly in the production of nitrate salts in water. In addition, nitrates and nitrites are found as natural components of certain vegetables and are used as food additives (Eisenberg et al., 1970). Nitrates in food and water, particularly when consumed by infants, can later be reduced to nitrites by the bacterial flora of the upper part of the gastrointestinal tract, which in turn convert the hemoglobin into methemoglobin (Knotek et al., 1964). Drugs containing organic nitrates can release nitrite ions in the body by an enzyme-catalyzed reaction with reduced glutathion, and perhaps by other mechanisms (Goodman and Gilman, 1970). Infant methemoglobinemia resulting from the consumption of water with high nitrate concentration was recognized first clinically and investigated by Comly in 1945. Since then, a large number of cases,including fatal ones, among infants consuming water with high concentrations of nitrates, have been reported throughout the world (Gruener and Shuval, 1970). The most susceptible population at risk are infants who consume nitrate-containing substances, especially to the age of 6 months ( Shuval and Gruener, 1972). Up until now only a few studies have been carried out on the effects of nitrates and nitrites on humans and animals and in general these chemicals have not been considered particularly toxic. (Drukery, 1962; Lehman, 1958). In fact, 200 ppm of sodium nitrite are allowed in most forms of processed meat products. As yet, little attention has been paid to the toxicological effects of nitrites on the CNS. 409 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
410
BEHROOZI
ET
AL.
Petukov and Ivanov (1970) h ave recently reported on psychophysiological changes in schoolchildren in regions of high nitrate concentration in drinking water. The objectives of the present study which is one of a series of studies on the toxicology of nitrites (Gruener and Shuval, 1972) were to investigate the longterm chronic effects of NaNO, administered in drinking water on the CNS and changes which it would cause in the brain electrical activity as indicated by EEG deviations. MATERIALS
AND
METHODS
Sixteen male albino rats of the Hebrew University’s ‘Sabra’ strain weighing initially 200 & 10 g each were used. Four monopolar ball-pointed silver electrodes were stereotaxically placed on the dura mater of the cortex in the left and right anterior and left and right posterior areas ( Nir et al., 1969). The electrodes were fixed in place with dental cement and later joined to a connector, cushioned on the skull. After the implantation of the electrodes, the animals were allowed to recuperate for 2-3 weeks before the recordings were made. Animals were divided into four groups, each group comprising four rats. Each rat was placed in a separate cage. All groups received plain tap water for a period of 2 weeks during which time recordings from all of them were taken every 3 days in order to serve as controls of their own for later comparisons. The three experimental groups received sodium nitrite in their drinking water for a period of 2 months, after which time the NaNO, was removed from their drinking water and the animals received plain tap water for an additional period up to 4%months. The four groups were as follows: Group Group Group Group
A Control Group received only plain tap water B Experimental Group received: 2000 mg/l NaNO? C Experimental Group received: 300 mg/l NaNOz 100 mgll NaN02 D Experimental Group received:
All the recordings were made under identical conditions and time. The room temperature varied between 19 and 22°C. During the recordings the animals were kept in a round metallic cage and permitted to move about freely while recordings were made and animals’ behavior could be observed. After connection of the EEG adaptor to the socket, the animals were allowed a period of X5-20 min for adjustment. The average length of recording was 65 min continuously. EEG recordings were taken every 3 days until the end of the experiment. An Alvar 8 channel electroencephalograph was used in this study. After each recording blood samples were taken from the tail of the rats for hemoglobin and methemoglobin determination ( Hegesh et al., 1970). The evaluation of EEG records was made by visual methods and wave group counting. RESULTS
Different concentrations of NaNO, in the drinking water revealed characteristic changes in the EEG pattern as well as methemoglobin formation and behavioral changes.
EFFECT
CONTROL R.A-L.A.
OF CHRONIC
EXPOSURE
TO SODIUM
IS
2 WEEKS
ey
411
KITRITE
WEEKS -+..--4~rvuy‘~,iic
~.-++.-p,-v
50 pv.llsec FK. low-up
Group
1. The EEG period (Group
of the control A Control).
group
remained
unchanged
during
the
., ‘.
(
3?& months
of fol-
A
Group A showed no changes in the brain electrical activity or in the behavior during the 3% month period of follow-up. There were also no changes in the pcrcentage of MHb. The mean percentage of MHb was 0.5% and varied between 0.0 and 0.8% which is normal in rats (Fig. 1). TABLE I
BETWEEN AMPLITUDE
MEAN
FREQUENCY
1 OF BXIWR~UND
RANGES .IND MICTH~ IN RATS CONCENTRATIONS OF Nah’O?
RIMIN COSSIJMING
I:LKCTHIC’.II, I)IFFP;KLNT
(;roup
I )
N:PiO:
100
mg ‘liter s.4 1.4s ::0-6tl 0 .-I 0 24 Ii I 0 7:; ::I)--17(1 I I (1 1; 7 .o 0 !)I :~o-xo 0 4’ 0. 10
412
BEHROOZI
ET
AL.
50 p11 FIG. 2. Change tion of the rhythm, waves (Croup B).
in background appearance
activity of spikes
sec.
,
after 2K weeks of NaN02 drinking showing and sharp waves, mixed with diffused theta
acceleraand delta
Group B This group was exposed to 2000 mg/liter NaNO, drinking solution which is equivalent to a daily dose of 280 mg/kg body weight of the rat per 24 hr. Changes in the brain electrical activity appeared from the fourth day of treatment. There appeared diffused spikes and sharp waves and the frequency of the background electrical activity gradually increased from a mean frequency cycles per second (X.f. 8.4 c/s) to Z.f. 11.3 c/s (see Table 1)) achieving maximum of frequency after 2 weeks of treatment. After 2 weeks, general paroxysmal outburst of high amplitude up to 240 ,uV sharp waves became apparent mixed with diffused theta waves (Figs. 2 and 3). During the 2 months of chronic exposure to NaNO, the EEG records were of similar pattern, Ranges of percentage of MetHb varied between 6.7 and 30.8% with average 12.16%. All the rats in this group were generally sedate when compared with the control group as well as with their own behavior before the treatment. During the electrical outbursts their behavior suddenly changed and the rats remained motionless. After the end of the outburst their motor activity returned to that of their previous pattern. During the electrical outbursts, there were no clinical convulsions or any collapse and the animals were awake.
FIG. 3. Appearance waves ( Group B ).
of general
paroxysmal
outbursts
of high
voltage,
theta,
delta,
and sharp
EFFECT
OF CHRONIC
EXPOSURE
TO SODIUM
413
XITRITE
50 yv.Tl sec. Frc. 4. delta, theta,
Background and sharp
activity still waves (Group
rapid
with
general
paroxysmal
,
outbursts
of high
voltage
B) .
Two of the four rats in the group showed pronounced cyanosis characteristics of methemoglobinemia with MetHb up to 30% and they seemed more sedate than the rest. In these rats the frequency of the occurrence of paroxysmal outbursts was greater and also of longer duration. During the 4% months of follow-up after withdrawal of NaNO, the EEG pattern was similar to that during the NaNO, treatment, i.e., the outbursts continued to appear and the background activity had a Z.f. 9.2 c/s and the MetHh range was between 0.3% and 0.7% ( Fig. 4). Group C This group was exposed to 300 mg/liter NaNO? drinking solution from which was received an equivalent daily dose of 42 mg/kg body weight of the rats per 24 hr. Here also changes appeared after 4 days of NaNO, treatment and the EEG recordings showed spikes and sharp waves, similar to Group B. The background activity appeared to be slower than that of their own control. Here, too, after 2 weeks of treatment there appeared general paroxysmal outbursts comprising slow and sharp waves with predominant slow waves of theta and delta bands. The frequency of the occurrence of these outbursts was less than in Group B, but the amplitudes were similar (Fig. 5).
50 p. FIG. 2 weeks
5. Appearance of slowed of NaNO, drinking (Group
background C).
activity,
diffused
theta
and
Ttsec
,
delta
waves
, after
414
BEHROOZI
ET
AL.
R&WATER R.A-LA.
FIG.
(Group
7”“““1pp-+
6. Background C).
activity
is still
slower
than
the control
and outbursts
continue
to appear
During the follow-up of 3% months after the withdrawal of NaN02, the EEG background activity had a %f. 8.0 c/s. General outbursts still contnued to appear (Fig. 6). The MetHb during the treatment ranged between 1.7 and 4.6% and after the withdrawal of NaNO,, it ranged between 0.3 and 0.7%. During the paroxysmal outbursts the animals in Group C behaved similarly to those in Group B. Group D This group was exposed to 100 mg/liter NaNOz drinking solution from which was received an equivalent daily dose of 14 mg/kg body weight of the rat per 24 hr. Here, too, changes in the EEG appeared after 4 days, and they seemed to be similar to those described in Group C, except that the frequency of the occurrence of outbursts was lower. During the return to plain tap water for a period of 2% months, slow background activity continued Tt.f. c/s 7.0. The general outbursts disappeared after 2 weeks but spikes and sharp waves were still recorded. The MetHb ranged between 0.8 and 2.4% (TI MHb% 1.1) and after the withdrawal of NaNO, the MetHb ranged between 0.3 and 0.6% (Fig. 8).
LA-L
.P !;~,,I~,~~~-~,~~~~~~~~~~.~~~~‘~~~~~~~’~ i
‘j, 50
FIG. 7. Background of high-voltage delta,
activity is slower than the control theta spikes, and sharp waves ( Group
with D).
appearance
p
Fsec
of general
(
outbursts
EFFECT
Ret.
R.A-L.A.
EXPOSURE
TO SODIUM
315
NITRITE
2WEEKS
---Ph++M~i
FIG. 8. ( Croup
WATER
OF CHRONIC
Slow
,-+---J4e--
background
activity
still
continues
with
diffused
spikes
and
sharp
waves
D ).
In general, their behavior was similar to Group A, but during the general outbursts they were similar to Group B. DISCUSSION
The results show that NaNO, has a strong effect on the EEG of the rats treated chronically with this substance. In the unfolding of the recordings obtained, it is noted that increased levels of NaNO, in drinking water led to: (1) more accentuated changes in the EEG; (2) increase in percentage of MetHb; (3) dccrease in general motor activity as observed visually. Changes appeared in the background EEG at the three concentration levels. 111rats exposed to 2000 mg/liter NaNO,, the background brain electrical activity appeared to be faster than the control group as well as the other two experimental groups, while at 300 mg/liter and 100 mg/liter NaNO,, the background electrical activity ‘became slightly slower than the control group. Spikes and sharp waves appeared in the EEG of all the rats in the experimental groups from the fourth day of the treatment and continued so during the whole length of the experiment as well as after their return to plain water. The described EEG changes might be due to brain anoxia caused by degenerative vascular changes in the brain. Hueper and Landsburg (1940) reported on the brain vascular degenerative changes and vacuolation in midbrain and brainstem with erythroltetranitrates and NaNO, and related the aforementioned phenomena to stagnant hypoxemia and hyperemia caused by vasodilatory effects of NaNO,. Another possible cause of hypoxia is due to nitrite inducing methemoglobinemia which results in the lowering of oxygen-carrying capacity of the blood. Garbuz ( 1971) in a brief communication has reported acceleration of the EEG rhythm in rabbits acutely treated with NaNO, and related this phenomenon to anoxic hypoxia which is caused by methemoglobinemia. In our present investigation, features which might suggest brain hypoxia expressed by the appearance of slowed background brain electrical activity in EEG were seen mainly at low concentrations of NaNO, in the drinking water of the rats with a mean of 1.1% MetHb in group D and 3.0% MetHb in group C. Such
416
BEHBOOZI
ET
AL.
levels are normal or nearly normal for rats and hypoxia would not be anticipated. It is noted that at the highest NaNOz dose of 2660 mglliter, when high MetHb levels were detected and anoxia m:ght log:cally be anticipated, the EEG showed rapid background activity not typical for hypoxic conditions. Our findings suggest that the highly disturbed EEG in our experimental groups after exposure to NaNO* might have been due also to irreversible toxic effects of the NaNO, itself on the CNS, even at relatively low levels of exposure. During the paroxysmal outbursts it was noted that the outbursts were diffused, symmetrical, synchronized and uniform. This would seem to suggest the outbursts are of centerencephalic origin. It is seen that NaNO, at different concentrations has different characteristic effects on the EEG. This might be due to biphasic effects of NaNO, on the EEG of the rats. Such phenomenon is known from the effects of other drugs such as pentobarbital. Changes in the brain electrical activity of the rats seem to be irreversible in that paroxysmal outbursts in Groups B and C, and spikes in Group D continue to appear even after the removal of NaNOz from the drinking water of the rats. It is essential to point out that it may still be premature to draw any conclusions from these findings as to the degree of risk that sodium nitrite may have for humans. Our findings of apparent irreversible brain damage at relatively low dose levels point to the potential risks that may be associated with the widespread human exposure to sodium nitrite, used as a food additive, or for that matter, nitrate in food and water which can be converted easily to nitrites. REFERENCES COMMLY, H. H. ( 1945). Cyanosis in infants caused by nitrates in well water, J. Amer. Med. Ass. 129, 112. DRUKERY, H. ( 1963). Arzneim. Forsch. 13, 320. EISENBERG, A., WISENBERG, E., AND SHWAL, H. I. (1970). ‘The Public Health Significance of Nitrate and Nitrite in Food Products,” Ministry of Health, Jerusalem. GARBUZ, A. M. ( 1971). Functional state of the central nervous system in “Symptomless” methemoglobinemia, Gig. Sanit. 36, 101-102. GOODMAN, L. S., AND GILMAN, A. ( 1970). “The Pharmacological Basis of Therapeutics.” 4th edition, pp. 754-770, The Macmillan Company, New York. GRUENER, N., AND SHUVAL, H. I. ( 1970). Health aspects of nitrates in drinking water, In “Developments in Water Quality Research,” pp. 8%106, Humphrey, Ann Arbor, MI. Stud ies on the toxicology of sodium nitrite. In CRUENER, N., AND SHUVAL, H. I. ( 1972). “Environmental Quality and Safety.” Vol. II, Academic Press, New York. HEGESH, E., GRUENER, N., COHEN, S., BOCHKOVESKY, R., AND SHWAL, H. I. ( 1970). A sensitive method for the determination of methemoglobin in blood. Clin. Chim. Acta 30, 679482. HUEPER, W. C., AND LANDSBERG, J. W. ( 1940). Experimental studies in cardiovascular pathology. I. Pathologic changes in the organs of rats produced by chronic nitrite poisoning, Arch. Pathol. 29, 633-648. ( 1964). Pathogenesis, incidence and possibilities of preventKNOTECK, Z., AND SCHMIDT, P. ing alimentary nitrate methemoglobinemia in infants. Pediatrics 34, 78. LEHMAN, A. J. ( 1958). Nitrates and nitrites in meat products. Ass. Food and Drug Oficiaki, U. S. 22, 136-138. ( 1969 ) . Changes in the electrical activity NIR, I., BEHROOZI, K., ASSAEL, M., AND IVRIANI, I. of the brain following pincalectomy. Neuroendocrinology 4, 122127. PETUKNOV, N. I., AND IVANOV, A. V. ( 1970). Investigation of certain psychophysiological
EFFECT
OF CHRONIC
EXPOSURE
TO SODIUM
SITRITE
41:
reactions in children suffering from methemoglobinemia due to nitrates in water. Hyg. Sanit. 35, 29-31. SHWAL, H. I., AND GRUENER, N. (1972). Epidemiological and toxicological aspects of nitrates and nitrites in the environment. Amer. J. Pub. Health (in press),
Effect
of Chronic
Exposure
to Sodium
on the Electroencephalogram K. BXHROOZI, Encironmstul School-Hebrew
S. ROBINSON,
Nitrite
of Rats
N. GHUENER,
AND H. I. SHUVAL
Health
L,ahoratory, Department of Medical Ecology, Hndassah Medical University, Jerusalem, Israel and Talbieh Psychiatric Hospital. Jerusa!em, Israel Received
February
2, 1972
Brain electrical changes were observed in rats chronically treated with NaNOl in drinking water ranging in concentration from 100 to 2000 mg/l. The brain electrical patterns did not return to normal after the withdrawal of NaNO?, even at the lowest exposure dose of 100 mg/liter which was equivalent to a daily dose of 14 mg/kg. Each concentration of NaN02 drinking solution presented a characteristic pictnrcb of its own, i.e., in the nnfolding electroencephalogram several types of changes brcame discernible tallying with the specific dosages of NaKO> drinking solutions consumed by the rats, having a strong common denominator. These brain electrical activity changes are discussed in this paper. The ever-increasing concentration of nitrogenous compounds in water, food, and air has become a great public health concern all over the world. Organic solids and liquid wastes and nitrogenous chemicals and fertilizers used extensively
in agriculture contribute greatly in the production of nitrate salts in water. In addition, nitrates and nitrites are found as natural components of certain vegetables and are used as food additives (Eisenberg et al., 1970). Nitrates in food and water, particularly when consumed by infants, can later be reduced to nitrites by the bacterial flora of the upper part of the gastrointestinal tract, which in turn convert the hemoglobin into methemoglobin (Knotek et al., 1964). Drugs containing organic nitrates can release nitrite ions in the body by an enzyme-catalyzed reaction with reduced glutathion, and perhaps by other mechanisms (Goodman and Gilman, 1970). Infant methemoglobinemia resulting from the consumption of water with high nitrate concentration was recognized first clinically and investigated by Comly in 1945. Since then, a large number of cases,including fatal ones, among infants consuming water with high concentrations of nitrates, have been reported throughout the world (Gruener and Shuval, 1970). The most susceptible population at risk are infants who consume nitrate-containing substances, especially to the age of 6 months ( Shuval and Gruener, 1972). Up until now only a few studies have been carried out on the effects of nitrates and nitrites on humans and animals and in general these chemicals have not been considered particularly toxic. (Drukery, 1962; Lehman, 1958). In fact, 200 ppm of sodium nitrite are allowed in most forms of processed meat products. As yet, little attention has been paid to the toxicological effects of nitrites on the CNS. 409 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
410
BEHROOZI
ET
AL.
Petukov and Ivanov (1970) h ave recently reported on psychophysiological changes in schoolchildren in regions of high nitrate concentration in drinking water. The objectives of the present study which is one of a series of studies on the toxicology of nitrites (Gruener and Shuval, 1972) were to investigate the longterm chronic effects of NaNO, administered in drinking water on the CNS and changes which it would cause in the brain electrical activity as indicated by EEG deviations. MATERIALS
AND
METHODS
Sixteen male albino rats of the Hebrew University’s ‘Sabra’ strain weighing initially 200 & 10 g each were used. Four monopolar ball-pointed silver electrodes were stereotaxically placed on the dura mater of the cortex in the left and right anterior and left and right posterior areas ( Nir et al., 1969). The electrodes were fixed in place with dental cement and later joined to a connector, cushioned on the skull. After the implantation of the electrodes, the animals were allowed to recuperate for 2-3 weeks before the recordings were made. Animals were divided into four groups, each group comprising four rats. Each rat was placed in a separate cage. All groups received plain tap water for a period of 2 weeks during which time recordings from all of them were taken every 3 days in order to serve as controls of their own for later comparisons. The three experimental groups received sodium nitrite in their drinking water for a period of 2 months, after which time the NaNO, was removed from their drinking water and the animals received plain tap water for an additional period up to 4%months. The four groups were as follows: Group Group Group Group
A Control Group received only plain tap water B Experimental Group received: 2000 mg/l NaNO? C Experimental Group received: 300 mg/l NaNOz 100 mgll NaN02 D Experimental Group received:
All the recordings were made under identical conditions and time. The room temperature varied between 19 and 22°C. During the recordings the animals were kept in a round metallic cage and permitted to move about freely while recordings were made and animals’ behavior could be observed. After connection of the EEG adaptor to the socket, the animals were allowed a period of X5-20 min for adjustment. The average length of recording was 65 min continuously. EEG recordings were taken every 3 days until the end of the experiment. An Alvar 8 channel electroencephalograph was used in this study. After each recording blood samples were taken from the tail of the rats for hemoglobin and methemoglobin determination ( Hegesh et al., 1970). The evaluation of EEG records was made by visual methods and wave group counting. RESULTS
Different concentrations of NaNO, in the drinking water revealed characteristic changes in the EEG pattern as well as methemoglobin formation and behavioral changes.
EFFECT
CONTROL R.A-L.A.
OF CHRONIC
EXPOSURE
TO SODIUM
IS
2 WEEKS
ey
411
KITRITE
WEEKS -+..--4~rvuy‘~,iic
~.-++.-p,-v
50 pv.llsec FK. low-up
Group
1. The EEG period (Group
of the control A Control).
group
remained
unchanged
during
the
., ‘.
(
3?& months
of fol-
A
Group A showed no changes in the brain electrical activity or in the behavior during the 3% month period of follow-up. There were also no changes in the pcrcentage of MHb. The mean percentage of MHb was 0.5% and varied between 0.0 and 0.8% which is normal in rats (Fig. 1). TABLE I
BETWEEN AMPLITUDE
MEAN
FREQUENCY
1 OF BXIWR~UND
RANGES .IND MICTH~ IN RATS CONCENTRATIONS OF Nah’O?
RIMIN COSSIJMING
I:LKCTHIC’.II, I)IFFP;KLNT
(;roup
I )
N:PiO:
100
mg ‘liter s.4 1.4s ::0-6tl 0 .-I 0 24 Ii I 0 7:; ::I)--17(1 I I (1 1; 7 .o 0 !)I :~o-xo 0 4’ 0. 10
412
BEHROOZI
ET
AL.
50 p11 FIG. 2. Change tion of the rhythm, waves (Croup B).
in background appearance
activity of spikes
sec.
,
after 2K weeks of NaN02 drinking showing and sharp waves, mixed with diffused theta
acceleraand delta
Group B This group was exposed to 2000 mg/liter NaNO, drinking solution which is equivalent to a daily dose of 280 mg/kg body weight of the rat per 24 hr. Changes in the brain electrical activity appeared from the fourth day of treatment. There appeared diffused spikes and sharp waves and the frequency of the background electrical activity gradually increased from a mean frequency cycles per second (X.f. 8.4 c/s) to Z.f. 11.3 c/s (see Table 1)) achieving maximum of frequency after 2 weeks of treatment. After 2 weeks, general paroxysmal outburst of high amplitude up to 240 ,uV sharp waves became apparent mixed with diffused theta waves (Figs. 2 and 3). During the 2 months of chronic exposure to NaNO, the EEG records were of similar pattern, Ranges of percentage of MetHb varied between 6.7 and 30.8% with average 12.16%. All the rats in this group were generally sedate when compared with the control group as well as with their own behavior before the treatment. During the electrical outbursts their behavior suddenly changed and the rats remained motionless. After the end of the outburst their motor activity returned to that of their previous pattern. During the electrical outbursts, there were no clinical convulsions or any collapse and the animals were awake.
FIG. 3. Appearance waves ( Group B ).
of general
paroxysmal
outbursts
of high
voltage,
theta,
delta,
and sharp
EFFECT
OF CHRONIC
EXPOSURE
TO SODIUM
413
XITRITE
50 yv.Tl sec. Frc. 4. delta, theta,
Background and sharp
activity still waves (Group
rapid
with
general
paroxysmal
,
outbursts
of high
voltage
B) .
Two of the four rats in the group showed pronounced cyanosis characteristics of methemoglobinemia with MetHb up to 30% and they seemed more sedate than the rest. In these rats the frequency of the occurrence of paroxysmal outbursts was greater and also of longer duration. During the 4% months of follow-up after withdrawal of NaNO, the EEG pattern was similar to that during the NaNO, treatment, i.e., the outbursts continued to appear and the background activity had a Z.f. 9.2 c/s and the MetHh range was between 0.3% and 0.7% ( Fig. 4). Group C This group was exposed to 300 mg/liter NaNO? drinking solution from which was received an equivalent daily dose of 42 mg/kg body weight of the rats per 24 hr. Here also changes appeared after 4 days of NaNO, treatment and the EEG recordings showed spikes and sharp waves, similar to Group B. The background activity appeared to be slower than that of their own control. Here, too, after 2 weeks of treatment there appeared general paroxysmal outbursts comprising slow and sharp waves with predominant slow waves of theta and delta bands. The frequency of the occurrence of these outbursts was less than in Group B, but the amplitudes were similar (Fig. 5).
50 p. FIG. 2 weeks
5. Appearance of slowed of NaNO, drinking (Group
background C).
activity,
diffused
theta
and
Ttsec
,
delta
waves
, after
414
BEHROOZI
ET
AL.
R&WATER R.A-LA.
FIG.
(Group
7”“““1pp-+
6. Background C).
activity
is still
slower
than
the control
and outbursts
continue
to appear
During the follow-up of 3% months after the withdrawal of NaN02, the EEG background activity had a %f. 8.0 c/s. General outbursts still contnued to appear (Fig. 6). The MetHb during the treatment ranged between 1.7 and 4.6% and after the withdrawal of NaNO,, it ranged between 0.3 and 0.7%. During the paroxysmal outbursts the animals in Group C behaved similarly to those in Group B. Group D This group was exposed to 100 mg/liter NaNOz drinking solution from which was received an equivalent daily dose of 14 mg/kg body weight of the rat per 24 hr. Here, too, changes in the EEG appeared after 4 days, and they seemed to be similar to those described in Group C, except that the frequency of the occurrence of outbursts was lower. During the return to plain tap water for a period of 2% months, slow background activity continued Tt.f. c/s 7.0. The general outbursts disappeared after 2 weeks but spikes and sharp waves were still recorded. The MetHb ranged between 0.8 and 2.4% (TI MHb% 1.1) and after the withdrawal of NaNO, the MetHb ranged between 0.3 and 0.6% (Fig. 8).
LA-L
.P !;~,,I~,~~~-~,~~~~~~~~~~.~~~~‘~~~~~~~’~ i
‘j, 50
FIG. 7. Background of high-voltage delta,
activity is slower than the control theta spikes, and sharp waves ( Group
with D).
appearance
p
Fsec
of general
(
outbursts
EFFECT
Ret.
R.A-L.A.
EXPOSURE
TO SODIUM
315
NITRITE
2WEEKS
---Ph++M~i
FIG. 8. ( Croup
WATER
OF CHRONIC
Slow
,-+---J4e--
background
activity
still
continues
with
diffused
spikes
and
sharp
waves
D ).
In general, their behavior was similar to Group A, but during the general outbursts they were similar to Group B. DISCUSSION
The results show that NaNO, has a strong effect on the EEG of the rats treated chronically with this substance. In the unfolding of the recordings obtained, it is noted that increased levels of NaNO, in drinking water led to: (1) more accentuated changes in the EEG; (2) increase in percentage of MetHb; (3) dccrease in general motor activity as observed visually. Changes appeared in the background EEG at the three concentration levels. 111rats exposed to 2000 mg/liter NaNO,, the background brain electrical activity appeared to be faster than the control group as well as the other two experimental groups, while at 300 mg/liter and 100 mg/liter NaNO,, the background electrical activity ‘became slightly slower than the control group. Spikes and sharp waves appeared in the EEG of all the rats in the experimental groups from the fourth day of the treatment and continued so during the whole length of the experiment as well as after their return to plain water. The described EEG changes might be due to brain anoxia caused by degenerative vascular changes in the brain. Hueper and Landsburg (1940) reported on the brain vascular degenerative changes and vacuolation in midbrain and brainstem with erythroltetranitrates and NaNO, and related the aforementioned phenomena to stagnant hypoxemia and hyperemia caused by vasodilatory effects of NaNO,. Another possible cause of hypoxia is due to nitrite inducing methemoglobinemia which results in the lowering of oxygen-carrying capacity of the blood. Garbuz ( 1971) in a brief communication has reported acceleration of the EEG rhythm in rabbits acutely treated with NaNO, and related this phenomenon to anoxic hypoxia which is caused by methemoglobinemia. In our present investigation, features which might suggest brain hypoxia expressed by the appearance of slowed background brain electrical activity in EEG were seen mainly at low concentrations of NaNO, in the drinking water of the rats with a mean of 1.1% MetHb in group D and 3.0% MetHb in group C. Such
416
BEHBOOZI
ET
AL.
levels are normal or nearly normal for rats and hypoxia would not be anticipated. It is noted that at the highest NaNOz dose of 2660 mglliter, when high MetHb levels were detected and anoxia m:ght log:cally be anticipated, the EEG showed rapid background activity not typical for hypoxic conditions. Our findings suggest that the highly disturbed EEG in our experimental groups after exposure to NaNO* might have been due also to irreversible toxic effects of the NaNO, itself on the CNS, even at relatively low levels of exposure. During the paroxysmal outbursts it was noted that the outbursts were diffused, symmetrical, synchronized and uniform. This would seem to suggest the outbursts are of centerencephalic origin. It is seen that NaNO, at different concentrations has different characteristic effects on the EEG. This might be due to biphasic effects of NaNO, on the EEG of the rats. Such phenomenon is known from the effects of other drugs such as pentobarbital. Changes in the brain electrical activity of the rats seem to be irreversible in that paroxysmal outbursts in Groups B and C, and spikes in Group D continue to appear even after the removal of NaNOz from the drinking water of the rats. It is essential to point out that it may still be premature to draw any conclusions from these findings as to the degree of risk that sodium nitrite may have for humans. Our findings of apparent irreversible brain damage at relatively low dose levels point to the potential risks that may be associated with the widespread human exposure to sodium nitrite, used as a food additive, or for that matter, nitrate in food and water which can be converted easily to nitrites. REFERENCES COMMLY, H. H. ( 1945). Cyanosis in infants caused by nitrates in well water, J. Amer. Med. Ass. 129, 112. DRUKERY, H. ( 1963). Arzneim. Forsch. 13, 320. EISENBERG, A., WISENBERG, E., AND SHWAL, H. I. (1970). ‘The Public Health Significance of Nitrate and Nitrite in Food Products,” Ministry of Health, Jerusalem. GARBUZ, A. M. ( 1971). Functional state of the central nervous system in “Symptomless” methemoglobinemia, Gig. Sanit. 36, 101-102. GOODMAN, L. S., AND GILMAN, A. ( 1970). “The Pharmacological Basis of Therapeutics.” 4th edition, pp. 754-770, The Macmillan Company, New York. GRUENER, N., AND SHUVAL, H. I. ( 1970). Health aspects of nitrates in drinking water, In “Developments in Water Quality Research,” pp. 8%106, Humphrey, Ann Arbor, MI. Stud ies on the toxicology of sodium nitrite. In CRUENER, N., AND SHUVAL, H. I. ( 1972). “Environmental Quality and Safety.” Vol. II, Academic Press, New York. HEGESH, E., GRUENER, N., COHEN, S., BOCHKOVESKY, R., AND SHWAL, H. I. ( 1970). A sensitive method for the determination of methemoglobin in blood. Clin. Chim. Acta 30, 679482. HUEPER, W. C., AND LANDSBERG, J. W. ( 1940). Experimental studies in cardiovascular pathology. I. Pathologic changes in the organs of rats produced by chronic nitrite poisoning, Arch. Pathol. 29, 633-648. ( 1964). Pathogenesis, incidence and possibilities of preventKNOTECK, Z., AND SCHMIDT, P. ing alimentary nitrate methemoglobinemia in infants. Pediatrics 34, 78. LEHMAN, A. J. ( 1958). Nitrates and nitrites in meat products. Ass. Food and Drug Oficiaki, U. S. 22, 136-138. ( 1969 ) . Changes in the electrical activity NIR, I., BEHROOZI, K., ASSAEL, M., AND IVRIANI, I. of the brain following pincalectomy. Neuroendocrinology 4, 122127. PETUKNOV, N. I., AND IVANOV, A. V. ( 1970). Investigation of certain psychophysiological
EFFECT
OF CHRONIC
EXPOSURE
TO SODIUM
SITRITE
41:
reactions in children suffering from methemoglobinemia due to nitrates in water. Hyg. Sanit. 35, 29-31. SHWAL, H. I., AND GRUENER, N. (1972). Epidemiological and toxicological aspects of nitrates and nitrites in the environment. Amer. J. Pub. Health (in press),