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test material. Exposure to 186 or 263 mg/m3 sulfur dust resulted in a response decrease of 7 and 17%, respectively. Since the breathing patterns of these mice appeared normal, with no apparent pause between inspiration and expiration (data not shown), the decrease in frequency did not reflect sensory irritation of the upper airway. At the highest exposure level (451 mg/rn3), a slight increase in respiratory rate was observed. Owe mouse in this group had a 2 4 % increase in respiratory rate and exhibited a breathing pattern indicative of pulmonary or deep lung irritation throughout the exposure period. The other three mice in this group showed normal breathing patterns. The eyes of all animals appeared normal during the exposure period. Upon removal from the chamber, two animals at 106 mg/m3 and three animals at 451 rng/m3 were observed with slight lacrimation. One day later, the eyes of d l mice appeared normal.

Discussion The present study was the first quantitative investigation of the irritation potentid of sulfur dust. Exposure levels ranged from 10 to 45 times the Occupational Exposure Limit of the Alberta Government, which is 10 mglm3, in an effort to generate a sensory irritation response. The average particle size, although larger than that considered respirable for mice, was considered small enough to be inhdable and to be deposited primarily in the nasal turbinates. Only a small portion of the sulfur dust was considered to have reached the lower airway as a result of the filtering efficiency of the rodent nasal passages. This unique distribution of deposited particles advantageous since the was conducted was evaluate sensory irritation which is a phenomenon of the nasal passages. The decreases that were observed in breathing rates in the low- and mid-dose groups were within normal limits and were V

not biologically significant. Furthermore, the animal breathing patterns were not indicative of sensory irritation. These results suggest that sulfur dust possesses little potential to stimulate the trigeminal nerve endings in the nasal passages to cause sensory irritation. Exposure to 451 mg/m3 sulfur dust resulted in one mouse exhibiting a change in breathing pattern that indicated deep lung, or pulmonary, irritation and resulted in a 2 4 % increase in breathing frequency. Given the 22-min exposure period and the high concentration of sulfur dust, it is plausible that a sufficient amount of sulfur dust reached the lower portions of the lung to produce an irritation response. Because of the limitations of the test system, further investigation of the lower airway response may be needed.

Acknowledgements The authors thank Mr. John Sigouein for his technical assistance and Ms. Patricia Fellin for the preparation of the manuscript. This work was performed under contract for the Canadian Petroleum Association. Alarie, Y. 1966. Irritating properties of airborne materials to the upper respiratory tract. Arch. Environ. Health, 13: 433-449. Alarie, Y. 1973. Sensory irritation by airborne chemicals. @R@ Crit. Rev. Toxicol. 2: 299-363. Alarie, Y. 198 1. Bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man. Food Cosmet. Toxicol. 19: 623 -626. Cauna, N., Hinderer, K. H., and Wentges, R. T. 1969. Sensory receptor organs of the human nasal respiratory mucosa. J . Anat. 124: 87 -209v Keele, C. A. 1962. The common chemical sense and its receptors. Arch. Int. Pharmacodyn. Ther. 139: 547 -557. National Institutes of Health. 1985. Guide for the care and use of laboratory animals. NIH Publication 85-23.

Effect of lithium chloride on ornithine decarboxylase activity in rat adrenal ALESSANDWA STWWMPFER, JERRYHSIAO,TEDPENG, A N D JAMESRICHARDS' Deparfmenf of Biochemis~ry,University of British Columbia, Vancouver, B. C . , Carlacia V6T B W5

Received March 2, 1992 STRUMPFER, A., HSIAO,J., BENG,T., and RICHARDS. J. 1992. Effect of lithium chloride on ornithine decarboxylase activity in rat adrenal. 78: 1293- 1296. The effects of lithium chloride on ornithine decarboxylase (ODC) activity were compared in the adrenal and kidney of control (saline treated) and prolactin-treated rats. ODC activity was decreased in kidney of both groups of animals, the magnitude of the effect of lithium in the hormone-treated group varying with the time of administering the lithium relative to prolactin. The response in the adrenal was quite different. Following treatment with LiC1, there was a gradual increase in ODC activity from a low of 16-35 pmol CO, 30 min-' mg protein-l in control animals to values 20- to 38-fold greater at 5 h. In rats treated simltaneously with LiCl and prolactin, ODC activity was greater at 5 h than that observed in animals receiving either compound alone, indicating that their effects were additive. When LiCl was given 4 h after prolactin, i.e., 1 h before sacrifice, OBC activity decreased to a very low level at 5 h, as in other tissues. The increase in ODC activity in the adrenal following LiCl is of the same magnitude as the changes observed in tissues stimulated to undergo alterations in proliferation. differentiation, or metabolic or membrane activity by hormones and other external stimuli. Key words: ornithine decarboxylase, rat adrenal, effects of lithium.

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lAuthor for correspondence. Printed in Canada i Imprim6 au Canada

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CAN. J. PHYSIOL. PHARMACOL. VOL. 70. 1992

STROMPPER, A., WSIAO,J., PENG,T., et ~ ~ I C H A RJ.D 199%. S, Effect of lithium chloride on ornithine decarboxylase activity in rat adrenal. 70 : 1293- 1296.. On a compare les effets du chlomre de lithium (ClLi) sur l'aetivite d'ornithine dCcarboxylase (OD@)dans la surrknale et le rein de rats trait& avec de la prolactine et de rats t h o i n s (traitts avec une solution saline). k'activitb ODC a augment6 dans le rein des deux groupes d'animaux, 19ampli$udede l'effet du lithium, chez le groupe trait6 avec l'hsrrnone, variant avec la durCe d'adrninistration du lithium par rapport h celBe de la prolactine. La rkponse a Ctb trks differente dans la surrknale. Aprks le traiternent au ClLi, l'activitC 8 D C a augment6 grriduellement de 10-35 pmol C 0 2 30 min-' . mg prodine-', chez les animaux tirnoins, h des valeurs de faeteurs 28-30 plus ClevCes h 5 h. Chez les rats trait& simultanement avec le ClLi et la prolactine, l'activitk ODC a kt6 plus forte h 5 h que celle observCe ehez les anirnaux recevant l'un ou l'autre composC uniquement, indiquant une additivite de leurs effets. Lorsque le ClLi a CtC administrt 4 h aprks la prolactine, c.-8-41. I h avant le sacrifice, l'activitk OBC a dirninut B un taux trks faible h 5 h, cornrne dans les autres tissus. %'augmentation de l'aetivitk ODC dans la surrenale, aprks l'adrninistration du ClLi, est du meme ordre de grandeur que les variations observ k s dans les tissus, stimulks par des hormones ou autres stimuli externes, pour subir des alterations au niveau de Iri prolif6ration, de la differenciation, du mCtabolisme et de I'activitC rnembranaire. Mots cl& : ornithine dCcarboxylase; surrtnale du rat; effets du lithium. [Traduit par la redaction]

The biochemical effects of lithium chloride have become important because of the success in using lithium to treat mental illness. While interest has been focussed on the effects sf lithium salts on the cellular signalling mechanisms used by neurohormsnes and neurstransrnitters, and on cellular protein phssphorylation patterns and gene expression in the central nervous system (Avissar et a&.1988; Brami et ak. 1991; Divish et ak. 1991; Gasebolt and Jope 1981), lithium has been shown to affect processes in other tissues as well. Effects on glycogen metabolism, Na-K ATPase activity, amins acid uptake, and mitogenic activity have been demonstrated in a variety of tissues (Volontk 1988). Recent studies suggest that lithium may also affect the production of pslyamines because of its negative effect on the activity of ornithine decarboxylase (EC 4.1. I. 17, ODC), the enzyme that is the major site of regulation of polyamine formation in mammalian cells (Janne ef aE. 1978; Pegg 1986). Lithium prevented the small, but essential, early increase of ODC in lectin-stimulated lymphocytes (Mustelin et a&.1986), and also prevented the rise of ODC in Ehslick ascites cells following dilution of cells in fresh serurncontaining medium (Matsui-Yuasa et aE. 1991). In prolactindependent Nb2 lymphoma cells, LiCl caused a large and rapid decrease in OBC activity when added to exponentially growing cells, and d s o at least partially inhibited the increase in 8 D C activity that foll~wsthe addition of the prolactin to stationary Nb2 cells (Richards et a&.1990). The administration of LiCl to intact mice was followed by a decrease in OBC activity in kidney, heart, and spleen (Adlercmtz er a&.1986). We observed similar effects in liver, kidney, and thymus of rats treated with LiCl. and extended the studies to include the effect of the salt on ODC activity in liver and kidney of rats treated with prolactin or dexamethasone (Richards et al. 1990). Following treatment sf rats with either hormone, there is the regular induction of 8 D C reported in many tissues, with an increase evident by 1.5 -2 h, which continues to a maximum at about 5 h and is followed by a decline to normal values by 8-9 h. When LiCl was given at hour 4 after the hormone, i.e., 1 h before sacrifice, the activity of ODC decreased dramatically (by 48-80%) in liver and kidney. When EiCl was given at the same time as the hormone, there was a much smaller effect ern OBC activity (10 -20 % decrease). During our studies on the effects of LiCl on ODC in tissues of control (saline treated) and hormone-treated rats, it was observed that the response of the enzyme in rat adrenal was different from that in other tissues. Further experiments on the ODC activity sf the adrenal are reported here.

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TABLE1. ODC activity (pmsl C 0 2 30 min- rng protein-') in rat adrenal and kidney after EiCl treatment *

Time after LiCl (h) Tissue

8

I

3

4

5

Kidney Adrenal

698+55 23 I0

522 61

472k28 149+%1

209 203

47f 18 574124

+

NOTE:Rats were treated with LiCl at time 0, and groups of three to five were sacrificed at the time shown. Data with f SEM were derived from three experiments. The other figures were obtained in a single experiment.

Materials and methods A n i m l studies Female Wistar rats (obtained from the Animal Care Centre, University of British Columbia) where used at 60 - 80 g body weight. Each experimental group contained three to five animals. Lithium cRloride and hormone were given by ip injection, according to the scheme for each experiment. Lithium chloride was given (5 pmollg body weight) in saline, and bovine prolactin (PRLB was given (480 pg) in saline at pH 8 - 8.5. Control animals received saline by ip injections. After sacrifice, the kidneys and adrenals were pooled for each group. Kidneys were homogenized with a Potter-EIvehern homogenizer in ice-cold buffer (50 rnM Hepes, 0. I mM EDTA, and 3 mM dithiothreitol, pH 7.3 -7.4). The adrenals were homogenized in the same buffer, using a small hand-driven glass homogenizer. The tissue homogenates were centrifuged at 20 000 X g for 20 min at 4"C, and the supernatant was used for the assay of ODC activity.

ODC activity Tissue extracts were assayed for ODC activity in triplicate by incubating aliquots of 100-250 p% with 260 mM ornithine (containing 0.25 yCi (I Ci = 37 GBq) of L-[I-%4C]ornithine),2dM) pM pyridoxal phosphate, 4 d v f dithiothreitol, and 100 yM EBTA in a total volume s f 400 pL. Radioactive C 0 2 production was measured as described previously (Richards et al. 1990). Hn separate experiments, it was found that the specific activity of ODC in tissues from individual animals treated with saline 5 h before sacrifice ranged from 600 to 800 pmol C 0 2 * 30 minu" mg protein-' in kidney and from %O to 35 pmol C 0 2 30 min-I mg protein-'in adrenal,

Assay of

Materials EiCl and other chemicals were obtained from Sigma Chemical Co., St. Louis, Mo. L-[l-14C]OrnitRine (50 -60 mCilrnmol) was purchased from the Amersham Corporation (BakviHle, Ont.). Bovine prolactin was supplied by the National Hormone and Pituitary Prsgram of National Institutes of Health in Bethesda, Md.

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TABLE2, Effect of LiCl on ODG activity of PRL-treated rats -

Time of treatment (h) Tissue

$RE

LiGl

Kidney

5

5 I

Adrenal

5 5 5

5

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5

Specific activity of OD6 (pmol CO, . 30 min-' . mg protein-')

5 I

NOTE:Each experimental group contained three to five animals. PWL was given at time 0, and all animals were sacrificed 5 k after the hormone treatment. LiCl was given either at time 0, for a 5-h treatment, or at 4 h for the l-h exposure. These results were obtained in three experiments.

Results A comparison sf the ODC activities in adrenal and kidney of rats at different times after LiCl treatment is shown in Table 1. In the kidney, the enzyme activity decreased from 698 pmol GO2 - 30 min-l . mg protein-' at time 0 to a value of 50 at hour 5. Over the same time period, the specific activity of 8 B C in the adrend increased gradually from 23 to 574 pmol C02 . 30 min-I mg protein-'. The increase was prevented by giving cycloheximide at the same time as LiGl (data not shown). The increase in ODC after LiCl was found o d y in the adrenal, and not in several other tissues, including liver, thymus, spleen, and heart. The results in Table 2 show the effects of LiCl on ODC activity in kidney and adrend when given at different times relative to treatment with PRL. As mentioned previously (Richards et ab. 1998), and included here for comparison, the activity in kidney is markedly decreased if lithium is given 4 h after the hormone, but the effect of lithium given with the hormone at time 0 is march less. The same acute reduction in ODC activity was observed also in the adrenal of PRL-treated rats given LiCl I h before sacrifice. However, in the adrenal, LiCl caused an increase in the activity when given simultaneously with PRE at time 0. The increase was roughly equivalent to the ODC induced by lithium in the absence of prolactin treat ment. The same additive effect was observed in the adrenal sf rats treated with growth hormone and lithium (data not shown). The effects s f EiCl are not due to direct interaction with the emyme. There was neither inhibition nor stimulation of 6 D C activity when LiCl was added directly to the assay tubes at concentrations up to 50 rnM (data not shown).

The effects of lithium chloride on ODC activity in rat adrenal are very similar to the pattern of induction of the enzyme in many other systems treated with a hormone, growth factor, carcinogen or tumor promoter, or other external stimulus (Sanne et nb. 1978; Russell 1985). The mechanism by which lithium produces this effect has not been investigated at the physiological or molecular level, except to show that the increase of ODC activity required enzyme synthesis. Changes in ODC mRNA or in enzyme stability might also be involved in this response. It is possible, too, that LiCl acts indirectly on the adrenal. It has been demonstrated that LiCl stimulates the secretion of adrenocorticotropin hormone (ACTH) in vtvo and in vttm (Vatid and Aiyar 1983; Reisine and Zatz 1987; Sugawara et ak. 1988). The response of ODC in the adrenal resembles

the induction of the enzyme in the adrenal of ACTH-treated rats observed in our laboratory and by others (Leuine et al. 1973, 1975; Richman et al. 1973). Further studies that focus on the physiological aspects of LiCl treatment (e.g., measuring ACTH or cort-ticoid levels) and on various stages of gene expression would provide information about the mechanism of action of LiCl. However, the purpose of this communication is to emphasize that the effect sf lithium on the adrenal seems unique, and could signify a potentially greater sverdl effect on that tissue. The effects of the salt on hormone induction of ODC in liver and kidney are quite small when given with the hormone, suggesting either that the effect is short lived or that the host can overcome or reverse the effect. In our previous study we found, too, that the pronounced effect on ODC in rapidly growing Nb2 cells was only partially sustained over longer periods sf time and cell division was not prevented (Richards eb a / . 1990). The effect of the LiCl in the adrenal is Barge and is more sustained than in other tissues. In fact, it resembles very closely the response of 6 D C in a tissue stimulated to undergo some change in rate of an important process such as cell proliferation, differentiation, or metabolic or membrane function.

Aekn~wledgemerats This work was supported by a grant from the Medical Research Council of Canada (J.F.R.). The authors thank Dr. Salvatore Raiti of the National Institute of Diabetes and Digestive and Kidney Diseases for a generous gift of bovine PWL. Adlercmtz, C., Rosengren, E., and Uvelius, B. 5986. Lithium lowers renal, cardiac and splenic ornithine deearboxylase activity in mice. Experientia, 42: 409. Avissnrr, S . , Schreiber, G., Danon, A . , and B e l w e r , R. H. 1988. Lithium inhibits adrenergic and cholinegic increases in GTP binding in cat cortex. Nature (London), 331: 4 0-442. Brami, B. A., Eeli, U.,and Hauser, G. 1991. Influence of lithiurn on second messenger accumulation in NG 108-15 cells, Biochern. Bisphys. Res. Gornmun. 174: 605 -612. Casebolt, T. L., and Jope, W. S. 1991. Effects of chronic lithium treatment on protein kinase c and cyclic AMP-dependent protein phosphorylation. Biol. Psychiatry, 29: 233-243. Divish, M. M., Sheftel. G., BoyIe, A . , Kalasapudi, V. D . , Papalos, D o F., and Lachman, H. M. 1991. Differential effect sf lithium on fos protooncogene expression mediated by receptor and postreceptor activators of protein kinase c and cyclic adenosine monophosphate: model for its antimanic action. J. Neurosci. k s . 28: 40-48.

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Janne, J., Piisti, H.,and Waina, A. 1978. Polyamines in rapid growth and cancer. Biochinn. Biophys. Acta, 473: 241 - 293. Levine, J. H., Nicholson, W. E., Liddle, G. W., and O~.eh,B. N. 1973. Stimulation of adrenal ornithine decarboxylase by adrenocortieotropin and growth hormone. Endocrinology (Baltimore), 82: 1089- 1095. Levirne, J. H., Nicholson, W. E., Peytremann, A., and Orth, D. N. 1975. The mechanism of ACTH stimulation s f adrenal ornithine decarboxylase activity. Endocrinology (Baltimore), 97: 134- 144. Matsui-Yuasa, I., Tsumta, D., Uernoto, M., Hasuma, T., Morisawa, S., and Otani, S. 1991. Control by treatment with lithium chloride of ornithine decarboxylase in Ehrlich aseites tumor cells. Biochem. Pharmacol. 41: 763 - 768. Mustelin, T e 9POSO,H., Iivanainen, A., and Andersson, L. C . 1986. myo-Inositol reverses Li'-induced inhibition of phosphoinositide turnsorerand ornithine decarboxylase induction during early lymphocyte activation. Eur. J. ImmunoB. 16: 859 -861. Pegg, A. E. 1984. Recent advances in the biochemistry of p l y amines in euhryotes. Biachem. J. 234: 249 -262. Reisine, T., and Zatz, M. 1987. Interactions among lithium, calcium, diacylglyeerides, and phorbol esters in the regulation of adrenocor-

ticotropin hormone release from AtT-28 cells. J. Neurochem. $9: 884 - 889. Richards, J. F., Fox, K . , Peng, T., Msiao, J., and Gout, P. W. 1990. Irnhibition of hormone-stimulated ornithine decarboxylase activity by lithium chloride. Life Sci. 47: 233 -240. Richman, R . , Bobbins, C., Voina, S . , Underwood, L., Mahafke, D., Gitelman, H. J., Van Wyk, J., and Ney, R. L. 1973. Regulation of adrenal ornithine decarboxylase by adrenocorticotrspic hormone and cyclic AMP. J . Clin. Inv. 52: 2807-2815. Russell, D. H. 1985. Onithine decarboxylase: a key regulatory enzyme in normal and neoplastic growth. Drug Metab. Rev. 16: 1-88. Sugawara, M., Hashimoto, K . , Hattori, T.. Takao, T., Suemam, S., and Oh,Z. 1988. Effects of lithium on the hypothalam-pituitaryadrenal axis. Endocrinol. Jpn. 35: 655 -663. Vatal, M., and Aiyar, A. S. 1983. Some aspects of csrticosterone metabolism in lithium treated rats. Chem. Biol. Interact. 45: 277 -282. Volontk, G. 1988. Lithium stimulates the binding of GTP to the membranes of PC22 cells cultured with nerve growth factor. Neurosci. Lett. 87: 127-132.