Effects of low-dose inhalation of three chlorinated aliphatic organic solvents on deoxyribonucleic acid in gerbil brain.

Effects of low-dose inhalation of three chlorinated aliphatic organic solvents on deoxyribonucleic acid in gerbil brain. Scand J Work Environ Health 13 (1987) 453-458. Young adult Mongolian gerbils (Meriones ungiculatusi were con tinuously exposed by inhalation to I, I,I-trichloroethane at the Swedish occupational exposure limit (70 ppm) , to methylene chloride at three times (210 ppm) the Swedish occupational exposure limit (70 ppm), and to perchloroethylene at three times (60 ppm) the Swedish occupational exposure limit (20 ppm), for three months, followed by a four-month postexposure solvent-free period. The concentratio ns of deoxy ribonucleic acid (DNA) were then determined in different regions of the gerbil central nervous system. It was observed that the DNA concentrations in several brain regions were decreased in the exposed animals. It was found that 1,I ,t -trichloroethane induced these alterations in many more brain areas at its Swedish occupational exposure limit than the other solvents studied at threefold thei r Swedish occupational ex posure limits. The results suggest that all the solvents decrease cell density by inhibiting the slow acqui si tion of DNA or by inducing cell death in some sensitive brain areas and that I ,I,I-trichloroethane should not be regarded as harmless as previously stated.

1,1,1-Tr ichloroethane (meth ylchloro form) , meth ylene chloride (dichloromethane), and per chloroethylene (tetr achloroeth ylene) are chlorina ted hydrocarbons that a re widely used in ind ustrial and con sumer product s. I,I,I-Trichloroethane has become increasingly popular during the last few decades becau se of its reput ed low degree o f to xicity (37). Ho wever , little is known of the neur oto xicity of the compound . Studies on workers in occu pat io na l settings have been made , but no ad verse ef fects on the cent ral ner vous syste m ha ve been o bserved a fter chronic exposure to 1,1,1trichloroethane (12,17,18); howev er , the exposure levels in these studies were relat ively low. Acute exposure of hum an s to I, I, I-trichlor oethane at 350 ppm has been show n to impa ir functio ns of the centra l nervous system in some ind ividual s (7). Lightheadedness, head ache and coordination problems, drow siness, a nesthesia, na rcosis, and even death may occur as a consequence of accidental human exposure to high solvent concentrations (4, 22,) 3 , 36). A few long-term stud ies on anima ls have been per formed, an d 1,1,1tri chloroeth ane has been reported to be well tolerated in several different species after lon g-term exposures at dif ferent solvent co ncentra tio ns (5,22,23).
Th e advers e effec ts associated with exposur e to meth ylene chlor ide are pr imaril y neurological and  (35,38). Central nervous system effect s are relat ed to the anesthetic properties of the solvent, and expo sure of humans to the so lvent at 500-1 000 ppm ha s been shown to cau se early sign s of central nervous system depression (35,42). In the occupational setting diffuse toxic brain damage, resulting in visual and auditory illusions , has been reported after chronic exposure to meth ylene chloride (39,40). Bilateral ternporallobe degeneration ha s been described in one individual a fter long-term exposure to high concentrations of th e solvent (2). Th e ad verse effects associated with exposure to perchloroethylene primarily involve the central nervous system. Exposure of humans to low levels of thi s solvent (100 ppm) ha s been shown to result in signs of central nervous system dep ression in some individuals (34), and it is aggra vated at higher concentrations (28). Central nervou s system symptoms, characteristic of the neurasthenic synd ro me, have been described after chronic exposure to high concentrations of perchloroethylene (8).
The purpose of this study was to compare the effects of chronic exposure to !,I,I -trich!oroethane, methylene chlor ide, and pcrchlorocthylene at low levels on the cell density of different distinct brain areas of the gerbil central nervou s system. The gerbils were exposed by continuo us inh alation to !,I,I-trichloroethane at 70 ppm , to methylene chloride at 210 ppm, and to per chloroethylene at 60 ppm for three months, followed by a post-exposure sol vent-free period of four months. Aft er thi s period the deoxyribonucleic acid (DNA) conc entration s were quantitatively analyzed as a measure of cell content in the various brain areas.

Animals
Twenty-four male and 24 female Mongolian gerbils (Meriones ungiculatus) were used. The initial mean weight of the male gerbils was 63.9 (SE 1.2) g and of the females 50.9 (SE 0.8) g. The male and female animals were housed separately in transparent plastic cages (45 x 24 x 15 em, four in each). The experimental and control animals were sex-matched littermates. Commercial laboratory rat chow (Astra Ewos, Sweden) and water was freely available. Sawdust was used as bedding. The environmental temperature was kept at 22 ± 2°e. Light supplementing daylight was automatically controlled for 12 h of daylight, with 30 min of twilight at dawn and evening.

Exposure
During the experimental run, the exposure was continuous. Commercial 1,1, l-trichloroethane, containing 5 % dioxane-free stabilizers (methyl ethyl ketone, methylene oxide, butylene epoxide, butanol, and nitromethane), methylene chloride, containing 0.3-0.5 % stabilizers (butylene oxide and antioxidants), and tetrachloroethylene, containing 0.01 % stabilizers, all of cleaning grade, were used (Billerud AB, Sweden). Each solvent was injected into a temperature-controlled glass-vaporizer (60°C), mixed with a small volume of air (20 IIh), and then diluted with clean air (1 OOOllh) to produce the desired concentration (70 ppm for 1,1,1trichloroethane, 210 ppm for methylene chloride, and 60 ppm for perchloroethylene). All air was filtered to remove oil and particles larger than 0.3~m. The concentration in the inhalation chambers was monitored with a Miran IA spectrophotometer fitted with a multipath gas cell. The long-term stability of the system permitted the concentration of the solvent to be held within 10 % of the decided concentration. Interruptions occurred only 1-2 h per week for the changing of water, bedding material, and food.
Four male and four female gerbils were exposed for each solvent concentration. Eight sex-matched littermates served as controls for each solvent concentration and were exposed to clean air (I 000 IImin) in chambers identical to those of the experimental animals. The exposure period was three months. Subsequently, the animals were removed from the inhalation chambers and kept free from exposure for a fourmonth period.

Preparation of tissue
After the solvent-free period, the gerbils were killed. The brains were removed from the skulls and separated from the spinal cords by transection at the distal end of the fourth ventricle. The olfactory bulbs were removed, and the brains were weighed. The cerebral cortices were dissected on ice and divided by transverse 454 cuts into three equally long parts, ie, frontal (frontal), middle (sensory-motor), and dorsal (occipital) cerebral cortex. The cerebellar hemispheres were divided at the position of the sulci intercrurales into anterior and posterior parts. The anterior (lobuli I-V) and posterior (lobuli VI-X) cerebellar vermis, as well as the brain stem (under the fourth ventricle) and the hippocampus, were also dissected. Tissue samples were weighed, quickly frozen on dry ice, and then stored in tightfitting boxes at -80°C until analyzed. Tissues were homogenized at 1:5 or 1:20 (weight: volume) in 0.024 M barbital buffer, pH 8.6, with 2.5 mM ethylene diaminetetraacetic acid and 1.0 mM 2-mercaptoethanol.

Analytical procedure
The protein concentrations in the homogenates were determined according to Lowry et al (14). Bovine serum albumin was used as the reference standard. Analyses of the DNA concentrations in the hornogenates were carried out according to Kissane & Robins (10). Salmon DNA was used as the reference standard.

Statistical evaluation
The nonparametric Fisher's permutation test for paired observations was used to test the differences observed between the control and the exposed groups (1).

Body and brain weights
No animal died during the exposure or the solvent-free periods. There were no significant differences in body weight between the exposed and control animals, either at the end of exposure or after the solvent-free period. Brain weights and the weights of the dissected brain regions were not significantly altered between the exposed and control animals.
Protein and deoxyribonucleic acid concentrations 1,1,1-Trichloroethane. The total protein concentrations per wet weight in the different brain regions studied were not significantly different after exposure to 1,1,1trichloroethane at 70 ppm. However, the DNA concentrations were significantly lower in the exposed animals in three different brain areas, ie, the posterior cerebellar hemisphere, the anterior cerebellar vermis, and the hippocampus (figure 1).
Methylene chloride. The total protein concentrations per wet weight in the different brain regions studied were not significantly changed after exposure to methylene chloride at 210 ppm, when compared to those of the controls. The DNA concentrations per wet weight were significantly decreased in the hippocampus after the exposure (figure 2). Perchloro ethy lene. The tot al pr otein con centrations per wet weight in the differ ent brain areas studied were not significa ntly changed after exposure to perchloroethylene at 60 ppm. The DNA conce ntra tio ns per wet weight were decreased in the fro nta l cerebral cortex after th e exposure ( figure 3).

Discussion
In this study, gerb ils were expo sed eith er to 1,1,1trichloroeth ane at the Swedish occup at ion al exposure limit (70 ppm ) or to meth ylene chloride at th ree times (210 ppm) the occupation al exposure limit (70 ppm) of the mean s, bla ck columns =values for con t rol ani mals, str ip ed columns =values for exposed animal s) or to pen:hloroethylene at three time s (60 ppm) the occupational expo sure limit (20 ppm) by co ntinuo us inhalation fo r three months. Pr eviou s experiment s with etha no l, perchlorocthylen e, meth ylene ch loride, and xylene con firm that thi s period o f time is suff icient to induce alt erations among di fferent cell populations in gerbil brain (24)(25)(26)(27). According to these studies, the anim als were subjected to a postexposure, solvent-free period of four months to perm it a n estimation of the lasting or permanent changes indu ced by the precedin g exposure.
In th is study no significant cha nges in bod y or brain weight s were found between the exposed and control anima ls. The concentrations of total proteins in the different brain areas were no t significantly affected by the exposure. Ho wever, exposure to 1,1, l -trichloroethane at the Swedish occupational exposure limit (70 ppm) induced lasting decreased DNA concentrations in three bra in areas , ie, the po sterior cerebellar hem isphere, the anterior cerebellar vermis , and the hippocampus, while exposure to methylene chloride and perchloroethylene at three times (210 or 60 ppm) their Swedi sh occupational expo sure limit s resulted in decrea sed DNA levels in the hippocampus and frontal cerebral cortex , respecti vely. On the assumption of a dip loid DNA content in the bra in cells and a metabolic stability of DNA in the adult br ain (9, 15), our result s indicate a decreased cell den sity in several different br ain region s that ma y be du e to a loss of cells. Thi s phenomenon might be ascribed to cell death of eg, ner ve or oligodendroglial cells, or the inhibition of the slow nonneuronal cell acquisit ion ph ase in the adult brain (II). Cellular event s occurrin g during changes of the normal en vironment of brain cells, especially with regard to neurotoxins , are complex. Our results demonstr ate, far beyond chance, that lon g-term exposure to these solvent s result s in altered DNA concentrations as a sign of neurotoxicity in different parts of the gerbil bra in . Although the mechani sm behind the neu rotoxicit y of the se so lvents is unkno wn, our result s clearly show that no correlation between the respective Swedish occupational exposur e limits and their effects could be determined.
I, I, l-Trichloroethane is metabolized to a negligible extent (30, 3 1), cont rary to meth ylene chloride (3,13), while perch loro eth ylene is metabolized to so me extent (21,32). Thus local biotransfo rmat ion o f the so lvents in the central ner vous system could not explai n why I , I, I-trichlor oethane seems to have greater ef fects than meth ylene chloride and perchloroethylene. However, in this cont ext , one ha s to consider that the central nervous system con ta ins a low content o f the cytochrome P-450-monoxygenase system (16) and that the brain levels of reactive metabolites might be low . With regard to meth ylene chloride, the gluta thione-dependent pathway of biotransformat ion present in the brain has to be co nside red (I), as well as the fact that meth ylene chlo ride-derived J4ca rbo n activity in brain mac romolecules has been demonstrated , a nd th is phenom enon might reflect metabolite bindin g (19,20). On the other ha nd , as I, I, I-tr ichloroethane is very lipid so luble (29) an d as high concentra tions o f the so lvent can reac h the central nervous system during expos ure (41), effects on br ain membranes a re prob able . Ho wever, perchl oroethylene is approximately five tim es more lipid so luble than 1,1 , I-tric hlor oetha ne and should reach even higher levels in the centra l ner vou s system (6). Lipid so lu bility could thu s not expla in our result s. Th e bio chemical alterations ind uced after chronic low-level exposure to I, I.l-trich loroethane in-dicates that this solvent cannot be regarded as harmless , as p r e vi ousl y sta te d (3 7 ), in co m pa ri so n to m ethyle ne c h loride and perchloroeth ylcn e.