ELSEVIER Brain Research 730 (1996) 47 51

BRAIN RESEARCH

Research report

Effect of low and high doses of nitrous oxide on preproenkephalin mRNA and its peptide methionine enkephalin levels in the hypothalamus

R.K. Agarwal ~''j, G. Kuge! b, A. Karuri a, A.R. Gwosdow ~, M.S.A. K u m a r ~"

~ I)el~artlncnt o[Anatomy and Celhdar Biology fu[ts School (~" Veterinary Medicine. 200 Westboro Road, N. Gra[hm, AIA 01536. (,%t h Department (~'Restoratit'e Dentistry. Tt{lt,~ School (?[Dental Medicim', Boston. MA, USA

~' Endocrine Unit. Mas,sachu~'ett,~ General Hospital. Boston, MA, USA

Accepted 2 April 1996

Abstract

The effect of exposure to nitrous oxide (N20) on the levels of preproenkephalin mRNA in the hypothalamus of rats was examined. In the first experiment, rats were exposed to 1000 ppm N20 lbr 8 h a day over 4 days. Compared with controls (which were exposed to air over the same duration), the N+O exposed animals exhibited significant elevations in preproenkephalin mRNA levels in the hypothala- mus. In a second experiment, rats were exposed to 60% N+O or air lbr 12, 24 and 48 h duration, and hypothalamic levels of preproenkephalin mRNA as well as methionine enkephalin were analyzed. Compared with controls, NeO exposed rats exhibited significant elevations in preproenkephalin mRNA levels. The levels on preproenkephalin mRNA were significantly higher after 48 h of N~O exposure than alter 12 h of N:O exposure. Similarly, the concentration of methionine enkephalin was significantly higher after 24 and 48 h of exposure to N,O than after exposure to 12 h of N,O or air. These results indicate that (a) exposure to N,O results in significant elevations in preproenkephalin mRNA levels, (b) the increased preproenkephalin mRNA levels appear to be proporlional to the concentration of N+O exposure as well as the duration of N,,O exposure, and (c) N,O-induced elevation in preproenkephalin mRNA levels is associated with corresponding increase in tissue concentrations of methionine enkephalin. In total, these results suggest that N,O selectively stimulates synthesis of methionine enkephalin in the diencephalic region of the brain.

Kevwords: Preproenkephalin; mRNA: Opioid: Nitrous oxide; Hypothalamus: Met-enkephalin

1. Introduct ion

Nitrous oxide (N,O) is a widely used dental anesthetic, but its mechanism of action at the level of central nervous system is poorly understood. In addition to its well-known anesthetic effects, N,O has been shown to have adverse

effects on the reproductive system. According to a recent report, chronic exposure to N+O is a significant public health problem among dental professionals [28]. A number of reports indicate that N+O probably acts via the endoge- nous opioid system to bring about its analgesic effects and

perhaps its reproductive effects. Nitrous oxide may either interfere with opioid receptor function or act on opioid

neurons directly in order to disrupt gonadotropin secretion or alter analgesic state. In-vitro studies indicate that N+O selectively alters the binding of naloxone [l]. Nitrous oxide

Corresponding author. Fax: + I (508) 839-2953. r Present address: National Eye Institute, National Institutes of Health,

Bethesda. MD 20892-1858, tlSA.

was also shown to decrease the I~-opiate receptor binding afl]nity for [3H]dihydromorphine [22], and N,O-induced analgesia was shown to be reversed b y ( - )-naloxone but

not by (+)-na loxone , indicating a stereospecificity at the opioid receptor site [20,25]. Furthermore, N,O-induced analgesia was also shown to be reversed by intracere- broventricular injection of anti-[3-endorphin in hot plate pain test [8]. Nitrous oxide-induced analgesia was also shown to be significantly reduced by relatively selective

K-opioid receptor antagonists [3,25-27]. Although N~O-in- duced analgesia seems to be K-mediated in the mouse abdominal constriction test, NzO-mediated analgesia in

other species and other tests appears to be mediated by different opioid receptors [18]. In addition, chronic expo- sure of rats and mice to NeO has been shown to induce tolerance to the analgesic effects of the gas and withdrawal symptoms when removed from the analgesic environment [2,12]. Development of tolerance to N,O may involve release of endogenous opioid peptides. In support of this idea, enkephalinase inhibition by phosphoramidon has been

(1006-8993/96/$15.00 Cop2~right ~+' 1996 Elsevier Science B.V. All rights reserved. /'1[ S0006-~9 ~) ~(q6}(10429 5

48 R.K. Agarwal et al. / Brain Research 730 (1996) 47- 51

shown to prevent the development of tolerance to N,O analgesia in rats [29]. Chronic exposure to N20 has also been shown to result in a decrease in brainstem opiate receptor density [21]. Acute exposure to N20 has been shown to elevate the levels of [3-endorphin in the medial basal hypothalamus and periaqueductal gray area of rats [30] and consistent increases in [3-endorphin release from the superfused rat basal hypothalamic cells attached to Cytodex beads [31]. Reports on the effects of N20 on the enkephalin system appear to be variable. For example, acute exposure of rats to N_~O was shown to either increase Met-enkephalin levels in the CNS areas [24] and in the cerebrospinal fluid samples from lateral ventricles [6,23], or lack any effect on Met-enkephalin levels in the CNS areas of rats [19]. Our previous work indicates that NEO disrupts estrous cyclicity in rats [14], which is associated with an increase in hypothalamic GnRH and increases in Met-enkephalin levels in discrete areas of brain [22].

Although the effects of N20 on opiate receptors and levels of some endogenous opioids (such as Met-enkepha- lin and 13-endorphin) are partially explored, an analysis of the effects of N20 simultaneously on the messenger RNA levels for preproenkephalin and its peptide product, me- thionine enkephalin, is lacking. We report the effects of low and high doses of N,O on preproenkephalin mRNA and Met-enkephalin levels within the hypothalamus.

2. Materials and methods

Adult male Sprague-Dawley rats, weighing 200-250 g, were procured from Charles River Company (Wihnington, MA). The animals were housed in an AALAC approved housing facility, and the experimental design for these studies was approved by the University Animal Research Committee, in accordance with NIH guidelines.

After an acclimatization period of 7 days, animals were randomly assigned to the following two experiments.

2.1. Experiment 1

Groups of rats (n = 6) were exposed to 1000 ppm of N20 in air (1000 ppm = 0.1%), or air inside an environ- mental chamber for 8 h a day for 4 days, as described before [14,15], The dose of N20 used for this experiment is designed to mimic a typical dental practice personnel exposure [13]. The concentration of N20 inside the envi- ronmental chamber was monitored by random gas sam- pling using a MIRAN 101 specific vapor analyzer (Fox- boro Corp., South Norwalk, CT). At the termination of experiment, rats were sacrificed by decapitation, and brains were rapidly removed and the preoptic and hypothalamic area (referred to from now on as 'hypothalamus') was dissected out, as described previously [15]. Tissue samples were immediately frozen on liquid nitrogen and stored under -80°C until processed for RNA extraction.

2.2. Experiment 2

Rats were exposed to 60% N20 mixed with 19% nitro- gen and 21% oxygen. In this experiment, we selected a subanesthetic dose of N20 that has been shown to be effective in stimulating the secretory activity of the opioid system [31], in order to correlate duration of exposure to the gas with changes in the Met-enkephalin neural system. As described previously, rats were exposed inside an envi- ronmental chamber. Control animals (n = 9) were exposed to forced air inside the environmental chamber. Rats were removed from the environmental chamber after 12 ( n = 9 rats), 24 (n = 9 rats) or 48 h (n = 9 rats) of continuous exposure to NeO. Corresponding number of control ani- mals were exposed to air inside the chamber for 12, 24 or 48 h. All the animals were sacrificed by decapitation, and the brains were removed rapidly. Preoptic-hypothalamic fragments were dissected out and kept frozen at -80°C until processed for extraction of total RNA (three rats from each exposure group). For estimation of Met-enkephalin levels, brains from the remaining six rats from each group were subjected to peptide extraction as described below.

2.3. Peptide extraction and radioimmunoassav ~/ Met-en- kephalin

Tissues were weighed and homogenized in 2 N acetic acid and processed as described in detail previously [17]. Briefly, our extraction methods include an initial acetic acid homogenization step followed by high-speed (10000 × g) pelleting. The supernatant was lyophilized and re-ex- tracted with a mixture of acetonitrile/chloroform, and the clear supernatant fraction freeze-dried. Reconstituted ex- tract was then oxidized with 1% H202. The oxidized tissue extract was assayed for methionine enkephalin sul- t'oxide by a specific radioimmunoassay (RIA) developed in our laboratory [16,17]. Since peptides containing methion- ine tends to form sulfoxides, and developing highly sensi- tive antibodies against sulfoxide forms of Met-enkephalin is easier than developing antibodies against native form of Met-enkephalin, we have developed a highly sensitive RIA for Met-enkephalin sulfoxide. Peptide data were analyzed by analysis of variance with Scheffe's post hoc compar- isons.

2.4. Preparation of RNA

Total RNA from hypothalamic fragments were ex- tracted by using RNAzol TM B RNA isolation kit (Cinna/Biotecx Laboratories, TX). The single-step RNA isolation by acid guanidium thiocyanate-phenol-chloro- form extraction method has been described in detail else- where [4,10]. The RNA extraction method contained less contaminating proteins, as judged by the 260/280 UV absorption ratios consistently greater than 1.8. and ranging from 1.84 to 2.1. Yield of total RNA was approximately

R.K. Agarwal et al. /' Brain Re.search 730 ~ 1996) 47 .'71 4c)

1.7 ± 0.03 I~g /mg tissue. Extracted RNA was denatured by the addition of a small aliquot of loading buffer (5{)% formamide, 65f formaldehyde in MOPS buffer, heated to 65°C for 10 min). Denatured RNA was loaded (with tracking dye: bromophenol b lue/xylene cyanaol) in 8->g aliquots with one well containing different molecular mass markers, and electrophoresis was carried out on a 6% formaldehyde gel (2 g agarose, 33.8 ml 37% formalin, 20 ml running buffer (final concentration 20 mM MOPS, 5 mM sodium acetate. 0.5 mM EDTA) and 144 ml diethyl pyrocarbonate-treated water). At the end of electrophoretic separation, RNA was transferred from the gel to a nylon membrane (Gene-Screen, Dupont) in an electro-transfer apparatus (IBI Instruments: using phosphate buffer). The nylon membrane was heated to 80°C for 2 h under vac- uum, sealed in a plastic bag and stored in a refligerator until the hybridization step.

2.5. P r e p a r a t i o n (71 ~'DNA probe

Rat preproenkephalin cDNA-plasmid preparation was kindly donated by Dr. R.D. Howells. The cDNA probe was prepared by cleavage of the plasmid pRPE-I with P~'ull, as described before [11]. The cleaved 435-base pair cDNA fragment (pRPE-I (base pairs 165-600)) [9] repre- sents a coding region of the mRNA. This fragment was labeled with [{,->-~P]dCTP (New England Nuclear) by random primer synthesis [5,7] using a random primer kit (Promega gene kit) to a final specific activity of 2 - 4 × 10 s c p m / > g . The reaction mixture was purified by prelimi- navy chromatography using a Sephadex G-50 column.

2.6. H v b r i d i : a t i o H

The nylon membrane with the transferred RNA was prehybridized under standard conditions in a sealed plastic bag. The prehybridization solution contained salmon sperm DNA, 50c/~ lk~rmamide, 5 × SSC/0 .05 M NaH2PO a, 1 Y, Denhardt's solution, and 1% SDS. Prehybridization was carried out at 42°C for 2 h. Random primed [32p]pRPE-1 was denatured (by boiling 8 -10 min and rapidly cooling) and specifically hybridized on the nylon membrane. Hy- bridization was carried out in 12 ml of hybridization solution (similar to prehybridization solution, but contain- ~ng the labeled eDNA probe at 2 -3 × 107 cpm) overnight at 42°C. First washing of the membrane was carried out at low stringency conditions (2 × SSC + 0.1% SDS) at room temperature of t0 rain each, 3 - 4 times. Final four wash- ings were carried out under high stringency conditions (0.1 × SSC + ().19~ SDS at 52°C, 30 rain each). Subse- quent autoradiography was performed at - 8 0 ° C with in- lensifying screen (Dupont Lightening Plus). The mem- branes were then stripped and re-hybridized with rat beta actin probe, hnage density (absorption units × ram: au × ram) on all the autoradiographs were determined by den-

L J I I

N20 Control

Fig. I. Norlhern blot analysis of preproenkephalin mRNA m the hypo thalamus of rats. Niin.)us oxide group consisled of rat> exposed to 1000 ppln of the gas for S h/day for 4 days. Note significanl increases in preproenkepllalin mRNA levels in N,O-exposcd rats

sitolnetric analyses (Pharmacia LKB [Iltroscan XL laser densitometer).

3. Results

Exposure to low levels of N~O ( 1000 ppm) resulted in marked increase in preproenkephalin mRNA levels in the hypothalamus compared to control animals (Fig. I). The two control animals exhibited basal levels of pre- proenkephalin mRNA levels (au × m m readings: 0.63 ± 0.15). The N,O exposed animals exhibited approximately a 2-fold increase in the levels of preproenkephalin mRNA levels (au × m m = 1.28 ± 0.15).

Northern blot analysis indicated an apparent duration- response in preproenkephalin mRNA levels in response to 6()~7~ N,O exposure (Fig. 2). The a u . m m readings on the autoradiographs are as follows: Controls 0.385 ± 0 .084:12 h exposure to N+O: 0.9{)3 __+ 0 .02 :24 h exposure to N,O: 1.11 ± 0 . 2 6 : 4 8 h exposure to N+O 1.29±0.05. Maxi- mum increase in preproenkephalin mRNA was seen after 48 h of continuous exposure to 609~ N~O. In all Northern analyses, stripping and rehybridization of the membranes with rat beta actin probe revealed no significant changes in 28S RNA among different groups of aninlais.

. . . . mgmMmmm

Control 12 hrs 24 hrs 48 hrs

Fig. 2. Northern blot analysis of preproenkephalin mRNA in tile hypo thalamus of rats. Rats ,.,,'ere exposed to 12. 24 and 4< v, h of N.O (l~/Y'Tc N,O. 21c7~ oxygen and 19cX nitrogen) or air. Noic a duration of N,O exposure effect on the intensity of preproenkephalin mRNA bands.

50 R.K. Agarwal et al. / Brain Research 730 (1996) 47-51

5000 m

4500 [ ] control "~

4000 [] ~2

3500

3000

~" 2500

"~ 2000

1500

lO00

500

0

Treatment Groups

Fig. 3. Effect of 24 h and 48 h duration of N20 exposure on hypothala- mic Met-enkephalin levels in the rat (n = 6 per group). Control animals were exposed to air, and experimental group of rats were exposed to 60% N?O. 21% oxygen and 19c~ nitrogen. Open stars indicate significant changes in Met-enkephalin levels (P < 0.05).

Continuous exposure to 12 h of 60% N20 exposure did not significantly change the Met-enkephalin levels in the hypothalamus from those of controls (results not shown). However, exposure to 60% N: O over 24 h and 48 h significantly increased the Met-enkephalin levels in the hypothalamus ( P < 0.01 ; Fig. 3).

4. Discussion

Met-enkephalin levels. This would result fi'om either an increase in Met-enkephalin levels or a decrease in release of Met-enkephalin. From the results of the present study, it appears likely the increased Met-enkephalin levels in N~O-exposed animals result from an increased synthesis of proenkephalin, as indicated by the increased pre- proenkephalin mRNA levels. It is also apparent from the results that although N20 exposure resulted in a multi-fold increase in preproenkephalin mRNA levels, the corre- sponding increase in hypothalamic concentrations of Met- enkephalin was modest. This may be due to the following: (a) an increased rate of release of Met-enkephalin from the termini; (b) decreased rate of conversion of pro-enkephalin into mature peptide, and /o r (c) decreased translation of the message into pro-peptide. In addition, several hours may be intercalated between an increase in mRNA levels and a corresponding increase in final peptide product, due to the time required for translational and post-translational events. This may be the reason why we did not observe an increase in peptide levels associated with an increase in pro-enkephalin mRNA levels after 12 h exposure to 60(~ N~O.

We have previously shown that sub-chronic exposure to N20 results in impaired reproductive status (as evidenced by constant proestrus) [14], reduced fertility rates [14] and a build-up of hypothalamic GnRH content [15], probably due to impairment of gonadotropin release. Results of the present study indicate that N20 exposure results in signifi- cant up-regulation of opioid neurons, as evidenced by increases in proenkephalin mRNA levels, which may be important in impaired functioning of GnRH neurons.

The results of this study indicate that subchronic expo- sure to low and high levels of N20 results in elevations in preproenkephalin mRNA levels in rat hypothalamus. There are no previous reports on the effects of N20 on mRNA levels of opioid peptides, although numerous reports exist on the selective effects of N20 on opioid peptides within the central nervous system [15,30,31]. It is significant to note that 1000 ppm exposure over 4 days resulted in approximately a 2-fold increases in preproenkephalin mRNA levels because in most dental offices without a scavenging system, the N20 levels have been shown to reach 1000 ppm [13]. These results underscore the impor- tance of an efficient scavenging system for N20 in dental practice. It is possible that the effects of low levels of N20 exposure is cumulative in nature, even though the gas exposure is intermittent. This may explain the significant increases in the levels of proenkephalin mRNA.

We have also correlated, for the first time, changes in Met-enkephalin levels with changes in preproenkephalin mRNA levels after exposure to N20. It is difficult to interpret changes in tissue levels of neuropeptides without additional information on either synthesis or secretion of the peptide. We have previously demonstrated that expo- sure to 30% N~O results in an increase in brainstem

Acknowledgements

The authors wish to acknowledge Dr. Daniel Kilpatrick, Worcester Foundation for Experimental Biology, for al- lowing free access to his laboratory facilities and for providing technical advice. Dr. Richard D. Howells of the University of Medicine and Dentistry of New Jersey is also acknowledged for granting us permission to use the rat preproenkephalin probe, pRPE-1. This project was partly supported by a grant from Hills Corporation.

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