Gasoline is the generic term for petroleum fuel used mainly for internal combustion engines. It is complex, volatile, and highly flammable and contains over 500 saturated or unsaturated hydrocarbons having from 3 to 12 carbon atoms. About 110 million people are exposed to gasoline constituents in the course of refueling at gasoline stations 1. Major toxic risk comes from breathing exhaust fumes and evaporative and refueling emissions rather than from occasional skin contact from spills 2. Nonetheless, after gasoline application to skin, a decrease in glutathione concentration, glutathione S -transferase activity, and lipid peroxidation was observed in liver and brain 3. Gasoline-induced neurotoxic effects such as ataxia, tremor, acute or subacute encephalopathic syndrome were ascribable to intentional use (gasoline sniffing) and not to occupational exposure. Unfortunately, gasoline sniffing has become an increasingly rising phenomenon in the poor societies as a means for cheap mood alteration 4.

Leaded gasoline contains a range of organolead compounds, especially tetraethyl lead, are difficult to eliminate from the CNS, and the injuries induced usually result in permanent neurological deficits that cause medical as well as socio-economical problems 5. Ethylene glycol is another gasoline constituent and a general fuel additive widely used in automobiles for its ability to absorb water and to prevent overheating or freezing. Ingestion of ethylene glycol produces not only central nervous system depression, but also cardiopulmonary complications, acute renal failure, and delayed neurological sequelae 6.

The change from leaded to unleaded gasoline, in fact, represents a transformation of a risk factor for the human population chronically exposed to urban air polluted by automobile smokes 7.

Oxygenates include substances such as methanol, ethanol, Methyl tertiary butyl ether (MTBE), ethyl tertiary butyl ether (ETBE), tertiary butyl alcohol (TBA), and tertiary amyl methyl ether (TAME) are used as antiknock agents in place of lead derivatives and as substitutes for high octane aromatics in fuel 89. Methanol are toxic alcohols can be used as a fuel either alone or as a gasoline additive. Methanol is well absorbed in humans following inhalation, ingestion, or cutaneous exposure; it produces a transient mild depression of the CNS with headache, vertigo, and vomiting. Ethanol induced an increase in the levels of norepinephrine in paternally exposed offspring. Serotonin levels were reduced in the cerebrum, and met-enkephalin levels were increased in all brain regions of offspring from both maternally and paternally exposed rats 10. Methyl tertiary butyl ether (MTBE) is an aliphatic ether, a volatile, colorless, and inflammable liquid, currently the most widely used oxygenate as an octane enhancer in gasoline blends. In subchronic and chronic toxicity studies, MTBE appears to have low systemic toxicity; the main findings observed in rats were depressant effects on the CNS, typical of similar ethers 1112. Tertiary amyl methyl ether (TAME) causes a significant but transient CNS depression akin to, but slightly more severe than, that resulting from MTBE exposure at the same levels 13. Methylcyclopentadienyl manganese tricarbonyl (MMT) is an effective octane enhancer. The primary combustion products of MMT are Mn phosphate, Mn sulfate, and a Mn phosphate/Mn sulfate mixture. Concerns have been raised that the combustion products of MMT containing Mn could cause neurological symptoms similar to Parkinson's disease in humans, even at low levels of exposure 1415. In rats, increase in brain Mn delivery was observed with inhalation exposure following Mn absorption from the pulmonary tract and direct transport of Mn to the central nervous system along the olfactory nerve 16. High dose manganese selectively targets dopaminergic neurons in the human basal ganglia 1718. Another study by Erikson et al 19 found that rats had significantly decreased striatal glutathione (GSH) levels following subchronic MnSO4 inhalation.

Alkylbenzenes, including toluene and xylene, occur naturally in small amounts in gasoline blends and standard gasoline formulations. These compounds are mainly absorbed by inhalation causing dysfunction in the CNS. The effects vary from severe neurological disorder (acute inhalation due to sniffing abuse) to deficits in neurobehavioural function in occupationally exposed groups. Neurobehavioural effects deriving from sub-acute exposure to toluene have been investigated in rats 20 and mice 21.

As Egypt is now in the process of transferring into the use of unleaded gasoline, this study employed the two types of gasoline currently used in Egypt and focused on comparing their impact. The study was designed to evaluate the effects of gasoline vapour inhalation on the brain monoamine neurotransmitters, lipid peroxidation, GSH, SOD, Na⁺, K⁺-ATPase and AChE. In addition to these biochemical analyses, the impact of gasoline inhalation on the aggressive behaviour was assessed.

Data in table 1 show that the rats exposed to unleaded gasoline exhibited a decrease in the content of norepinephrine of the cerebral cortex in comparison with the control group. On the other hand, no statistically meaningful change was detected between the leaded and unleaded inhaled groups. Moreover, the content of dopamine was lower than that in the control or leaded gasoline groups. On the other hand, no statistical difference was found between the control and the leaded groups. Serotonin content was not affected in all the three experimental groups when comparison was made between each other.

In the hypothalamus, data in table 2 reveal that the norepinephrine, dopamine and serotonin in the group exposed to the unleaded gasoline were lower than those of the control group. Moreover, norepinephrine in this group was less than that in the leaded gasoline group and the dopamine was higher than that in the leaded gasoline inhaled group. Exposure to leaded gasoline induced a significant decrease in both dopamine and serotonin in comparison with the control group. Moreover, norepinephrine was unaffected in this group as compared with the corresponding values of the control.

Data presented in tables 3 show that the rats exposed to the two types of vapours encountered a significant decrease in the norepinephrine, dopamine and serotonin contents of the hippocampus as compared with the control group. On other hand, no significant changes were found in norepinephrine and serotonin between the leaded or the unleaded gasoline, but dopamine level was lower in the unleaded gasoline than leaded gasoline groups.

As shown in table 4, norepinephrine and dopamine levels were not significantly changed in the group inhaled the unleaded gasoline in comparison with the control group, on the other hand, serotonin was lower than the other two groups and dopamine was less than that in the leaded gasoline group. Serotonin content of the cerebellum was lower in the leaded gasoline group than control group, but the dopamine and norepinephrine were elevated above the control level.

Data in table 5 reveal that Na⁺, K⁺-ATPase activity and total protein content in the two groups exposed to gasoline were lower than the control, although no significant difference was found between each other. The rats exposed to the unleaded gasoline had a significantly lower acetylcholinesterase activity as compared with the control or leaded group. No statistical changes were detected in the acetylcholinesterase activity of the leaded group as compared with the control.

Table 6 shows that the rats exposed to the unleaded gasoline had a significant increase in lipid peroxidation in comparison with either the leaded gasoline or the control groups, but no statistical changes were found between the groups inhaled the leaded gasoline and the control. The GSH content was lower in both the leaded and unleaded gasoline groups than the control group, but GSH in the unleaded group was higher than in the leaded group. The unleaded gasoline exposed group exhibited a significant decrease in superoxide dismutase activity in comparison with either the leaded gasoline or the control groups. No statistical differences were found between the leaded and the control groups.

Table 7, 8 shows that the rats exposed to the leaded or the unleaded gasoline expressed a higher number of aggressive events as assessed by tooth chattering, threat posture, leaping and biting as well as boxing position in comparison with those of the control. On the other hand, in the duration of tooth chattering and threat posture were higher than those in the control group. No significant difference was observed between the leaded and unleaded gasoline.

The present study revealed that rats exposed to the unleaded-gasoline had significantly elevated levels of lipid peroxides and a decrease in the superoxide dismustase activity in the brain cortex region in comparison with the control or with the group exposed to the leaded gasoline. Lipid peroxidation is a marker of oxidative stress that facilitates the evaluation of the damage caused by free radicals on membrane lipids 3435. On the other hand, GSH was lower in either the leaded gasoline or the unleaded gasoline than the control group. Correlative study by Raza 3 revealed that after gasoline application to skin, a decrease in glutathione concentration, glutathione S -transferase activity, and lipid peroxidation was observed in liver and brain. Exposure to toluene, one of the gasoline constituents, both in vivo and in vitro, leads to reactive oxygen species formation in many tissues including brain tissue 363738. The study of Calderón-Guzmán et al 39 revealed that the aldehydes resulting from the oxidation of the methyl groups in the aromatic compounds of toluene and cresols play an important role in the cell-damage process due to their lipophilic properties. A similar result indicated that exposure to low concentrations of toluene leads to persistent effects on cognitive, neurological, and brain-structural properties in the rat 40. On the other hand, the results derived from the present trial indicate that Na⁺, K⁺-ATPase activity and total protein content in the leaded or unleaded gasoline groups were lower than those of the control group. The inhibition of the plasma membrane Na⁺, K⁺-ATPase might have been attributed to a direct effect of the solvents and alcohols in the gasoline on the membrane causing dissolving the its bilayers. The most outstanding are toluene (highly lipophylic) acts on membrane lipids and the effect is specifically on phospholipids and on the other hand the organic solvents act on membrane proteins 344142. Also as the functional hydroxyl group (OH^-) moves away from the functional methyl group (CH₃) attached to the aromatic ring of the solvents, the biogenic amines, lipid peroxidation, and Na⁺, K⁺-ATPase and total ATPase enzyme activity tend to suffer important changes, indicating that the stearic impediment effect and the inductive effect produced at the aromatic ring of the solvents play an important role in the response of biogenic amine metabolism, together with the enzymes and lipid peroxidation of animal brain membranes 39. Other study by Castilla et al 43 reported a reduction of the enzyme Na⁺, K⁺-ATPase in brain membranes when high doses of toluene vapour (30,000 ppm) were administered subchronically to rats, but there was an increased Na⁺, K⁺-ATPase activity during acute exposure. In agreement of this line of reasoning is the study of Gutteridge and Halliwell 44 showing that Na⁺, K⁺-ATPase reduces its activity up to 50% due to oxidative stress. The present findings show that the rats exhibited a decrease in the activity of AChE in the unleaded group in comparison with either the control or the leaded groups. In addition to its function in degrading acetylcholine and modulation of neural function; AChE as a structural protein appears to play an important role in axonal outgrowth 45. Correlative study by Capo et al 46 on ethylene glycol, which is consider one of the main constitutes of gasoline, demonstrated various types of neuronal toxicities after ethylene glycol intoxication. The mechanism could possibly be related to oxidation of cellular macromolecules or to relative reduction in acetylcholinesterase-containing neurons, thus potentiating cholinergic effects. In the present study, no statistical differences were detected among the serotonin levels of the cerebral cortex in the three groups; the control, the leaded and the unleaded. However, in the hypothalamus, hippocampus, cerebellum, the serotonin level was lower in the groups exposed to either type of gasoline as compared to the control group. The serotonin system originates in Raphe nucleus in brain stem and projects with extensive connections to the hypothalamus, cerebellum, and cereberum 47 and plays an important role in regulation of behaviour, mood, appetite, anxiety and sleep 48. The rats exposed to unleaded gasoline had a significant decrease in the dopamine of the cerebral cortex as compared with the other two groups, but in the hypothalamus and hippocampus dopamine was lower in either the leaded or unleaded group as compared with the control. On the contrary, dopamine was elevated in the cerebellum of leaded exposed group above both the unleaded gasoline and control. The fluctuations in the levels of dopamine in different brain areas may be related to the effects MMT which is an organic manganese (Mn) compound added to unleaded gasoline and the mechanisms of Mn neurotoxicity has focused on the oxidative properties of Mn and its interactions with the dopaminergic system, including biphasic increases and decreases in dopamine levels associated with Mn exposure 49. Also, Prabhakaran et al 50 suggested a new underlying mechanism in which Mn interaction with cellular DA to potentiate ROS activates NF-B as well as iNOS signaling, and all of which are involved in promoting dopaminergic cell death. On the other hand, another gasoline constituent; ethylene glycol was found to target the dopaminergic neurons in the basal ganglia thereby inducing serious intoxication manifested as focal inflammation and necrosis in the basal ganglia areas of the brain and these effects are associated with Parkinsonism 51. Toluene, another gasoline constituent, was reported to induce perturbation in the nigrostriatal tract responsible for the coordination of voluntary movement 52. The cell bodies of the dopamine system are located in the substabtia nigra of midbrain and project to the striatum of the basal ganglia to coordinate skeletal muscle contraction, moreover, DA innervations reaches out to the hypothalamus to regulate secretion of TRH and prolactin secretion, and then projects to the brain limbic system to modulate motivations and emotions 47. So, gasoline induced impairment of the DA system would result in serious impacts on the neural control of voluntary locomotion and would affect several behavioural aspects.

In the present study, the norepinephrine level in the cerebral cortex and hypothalamus of the unleaded group was less than that in the control. Also norepinephrine was reduced in the hippocampus of both unleaded and leaded gasoline groups in comparison with the control. In the cerebellum, norepinephrine level was higher in the leaded group as compared with the control group. It was reported that volatile solvents such as benzene, a main constituent of gasoline fuel, seems to interact with the synthesis and catabolism of catecholamines and serotonin in the brain, which might explain the neurotoxic effects of these solvents 53. In this regard, deficiencies of serotonin or other monoamine neurotransmitters such as dopamine and norepinephrine are linked with depression 5455. In reference to the present results of the behavioural tests, the rats exposed to either the unleaded or the leaded gasoline exhibited a significantly higher rate of anxiety and emotionality in comparison with the control animals. Although information related to the effect of gasoline inhalation on behavioural aspects is extremely scarce, yet, it is possible to interpret these results in the light of the effects of gasoline constituents. Another study demonstrated that lead exposure enhances predatory aggression in the cat and provide experimental support for a causal relationship between lead exposure and aggressive behaviour in humans 56. This was concomitant with deficiency in serotonin that plays an important role to counteract the aggressive behaviour 5758. A correlative study by Perreault et al 59 found that aggressive male coral reef fish had lower relative serotonergic activity than less aggressive males, and treatment with the serotonin reuptake inhibitor antidepressant drug flouxotine reduce the territorial aggressive behaviour. It was postulated that serotonin helps counterbalance the tendency of two other major neurotransmitters in the brain dopamine and noradrenaline to encourage overarousal, fear, anger, tension, aggression, violence, obsessive-compulsive actions, overeating, anxiety and sleep disturbances 60.

The present results revealed that gasoline inhalation induced significant fluctuations in the levels of the monoamine neurotransmitters in the cerebral cortex, hypothalamus, hippocampus and cerebellum. The group exposed to the unleaded gasoline exhibited an increase in lipid peroxidation and a decrease in AChE and superoxide dismutase. The physiological impairments were accompanied with a higher tendency towards aggressive behaviour as a consequence to gasoline inhalation.

Na⁺, K⁺-ATPase: total adenosine triphosphatase; SOD: superoxide dismutase; AChE: acetylcholinesterase; GSH: reduced glutathione; TBARS: lipid peroxidation; DA: dopamine; NE: norepinephrine; 5-HT: serotonin; CNS: central nervous system; ROS: reactive oxygen species; MMT: methylcyclopentadienyl manganese tricarbonyl; TAME: Tertiary amyl methyl ether; MTBE: Methyl tertiary butyl ether; ETBE: ethyl tertiary butyl ether; TBA: tertiary butyl alcohol.

The author declares that she has no competing interests.