A protein kinase C inhibitor attenuates cyanide toxicity in vivo

Toxicology 100 (1995) 129-137

A protein kinase C inhibitor Edward

U. Maduh”“,

aBiochemical Pharmacology

attenuates

cyanide toxicity in vivo *

Eric W. Nealleya, Huafu Steven I. Baskin*”

Songb, Paul C. Wangb,

Branch, Pharmacology Division, United States Army Medical Research Institute Aberdeen Proving Ground, MD 21010-5425, USA bHoward University. Washington. DC. USA

of Chemical Defense.

Received I5 November 1994; accepted 6 February 1995

Abstract We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, I-(S-isoquinolinesulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mgkg, in brain of anesthetized male micropig@ (6-10 kg). Brain high energy phosphates were analyzed using a “P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Tesla horizontal bore magnet. H-7, 1 mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN-). Prior to NaCN, H-7, or H-7 + CN- administration, baseline “P resonance spectra of I-min duration were acquired for 5- 10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 mitt, H-7 did not affect baseline cell energy profile as reflected by the 3’P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/S of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN- antagonism, underscoring the role of PKC in cyanide intoxication.

Ke~~orcis: Protein

kinase C; PKC inhibitors;

Hypoxia;

*The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the US Army or Department of Defense. In conducting

the research described in this report, the investigators adhered to ‘The Guide for the Care and Use of Laboratory Animals,’ NIH publication No. 86-23. * Corresponding author: Commander, USAMRICD, Bldg

E-3100, Attn: MCMR-UV-PB/Dr.

Baskin, Aberdeen Proving

Ground, MD 21010-5425. Telefax: (410) 671 1960. ’ Allsig Health and Toxicology, Washington, DC, USA.

Piperazines;

Cyanide

antidotes;

Mediators

1. Introduction

A primary

molecular

target

cytochrome oxidase (aa,), the the though (Way,

mitochondrial electron other enzyme systems 1984). By inhibiting

for cyanide action is terminal oxidase of transport chain, may also be affected

cellular respiration, blocks tissue utilization of oxygen leading to the inhibition of mitochondrial energy metabo-

cyanide

0 1995 Elscvier Science Ireland Ltd. All rights reserved 0300-483~95/509.50 SSDI 0300-483X(95)03078-T

130

E. U. Maduh et al. /Toxicology

lism and, thus, the synthesis of adenosine triphosphate (ATP) via oxidative phosphorylation (Keilin, 1929; Schubert and Brill, 1968). In acute exposure, such as by inhalation or by intravenous (i.v.) injection, the metabolic actions of cyanide are rapid: signs of toxicity appear in lo-30 s and fatality occurs within 2-12 min (Ballantyne, 1987). In addition to supportive care, the treatment plan for cyanide intoxication has utilized detoxification strategies employing (in the USA) a combination of sodium nitrite and thiosulfate (Way, 1984). Sodium nitrite stimulates methemoglobin formation, which has a greater affinity for the cyanide anion (CN-) than hemoglobin does, and binds CN- to form cyanmethemoglobin. This series of reactions, by sequestering the toxic CNin the form of cyanmethemoglobin, reduces the amount of CN- available to bind cytochrome oxidase, which results in the formation of CN-cytochrome oxidase complex (Way et al., 1984). Sodium thiosulfate serves as a sulphur donor for the enzymatic conversion of CN- to the less toxic thiocyanate (SCN-) by sulfurtransferases, e.g. rhodanese and 3-mercaptopyruvate sulfurtransferase (Wing et al., 1992; Maduh and Baskin, 1994). However, this and other antidotal approaches to cyanide therapy have been questioned (Way, 1984; Scharf et al., 1991) partly because nitrites can pose potentially life-threatening cardiovascular complications (Weiss and Ellis, 1933; Ivankovich et al., 1980; Baskin et al., 1992) and the formation of methemoglobin does not always correlate clinically or experimentally with treatment outcome or efficacy (Kruszyna et al., 1982; van Heijst et al., 1987). Furthermore, clinically controlling methemoglobin levels to maintain a desirable therapeutic blood profile in acute cyanide emergency can be very problematic because excessive methemoglobin disrupts the delivery of O2 to tissues and has been associated with death from hypoxia (van Heijst et al., 1987). Thiosulfate, on the other hand, when used alone as a cyanide antidote is not as effective as when it is used in combination with nitrites (Way et al., 1984; Isom and Johnson, 1987; Baskin et al., 1992). Even when used in combination with nitrites, very large doses of thiosulfate need to be administered parenterally, thereby raising the risk

100 (1995)

129-137

of ionic imbalance. Thiosulfate use also presents a perplexing question as to the cellular basis for its efficacy since its extramitochondrial site of action has not been reconciled with the localization of rhodanese which is primarily in the mitochondrion (Isom and Johnson, 1987). In view of these clinical problems, the development of novel pharmaco-logical approaches to rational anticyanide therapy would be justified. The characteristic mitochondrial inhibition, histotoxic hypoxia, and loss of metabolic energy ehcited by cyanide suggest that preservation of cellular bioenergetic parameters may be a useful strategy for counteracting its effects. Pharmacologic inhibition of PKC has been found to protect against cellular injury and tissue death associated with experimental models of hypoxia/ischemia (Saitoh et al., 1991; Sahai et al., 1994), a condition which also biochemically exhibits reductions in cellular energy metabolism similar to that seen in cyanide toxicity (Ballantyne, 1987; Maduh et al., 1991). In addition to a profound impairment of phosphorous energy metabolism, cerebral hypoxia or ischemia activates PKC, and pharmacological treatments aimed at limiting PKC activation also limit the degree of cell injury and tissue death arising from the hypoxic/ischemic insults (Madden et al., 1991; Louis et al., 1993). Cyanide toxicity is frequently used to model chemical hypoxia (Ahlemeyer and Krieglstein, 1989; Huang and Gibson, 1989) and as recently proposed, (Maduh et al., 1993) cyanide depletion of cellular energy may lead to PKC activation through the Ca2+ and phospholipid mechanisms (Nishizuka, 1992; Sahai, 1994). Thus, PKC-inhibiting drugs (Hidaka and Kobayashi, 1992) may also protect against cyanide intoxication. We have, therefore, hypothesized that pharmacologic inhibition of PKC may counteract cyanide-induced reductions in brain high energy phosphates. The pig has been suggested as a viable model for studying cyanide toxicity because its physiological and toxicological responses are similar to those of humans (Vick et al., 1990). In this study, we have examined the effects of the PKC inhibitor I-(S-isoquinolinesulfonyl)-2methylpiperazine, H-7, (Kawamoto and Hidaka,

E. U. Maduh

et al. / Toxicology

1984) on cellular energy depletion caused by sodium cyanide (NaCN). 31P nuclear magnetic resonance (NMR) spectroscopy (Chance, 1989) was used to analyze the time-course changes in high energy phosphate metabolism. Indices such as phosphocreatine (PCr), adenosine triphosphate (y-, CX-, fl-ATP, (r-ATP refers to the o-phosphate in ATP and the signal it generates), and other phosphorous compounds, such as phosphomonoester (PME), inorganic phosphate (Pi) and phosphodiester (PDE), were examined in the brain of miniature swine before and after i.v. injections of NaCN with or without H-7 pretreatment. 2. Materials and methods 2.1. Animals All data presented were acquired from adult male Yucatan micropigs@ (6-10 kg, n = 5). The animals were purchased from Charles River Laboratories (Wilmington, MA) and housed in individual (4 x 6 ft) pens with aluminium floors and access to tap water ad libitum. Pig chow (Purina 5084, Purina Mills, Inc. Richmond, IN) was provided twice a day. The cages were placed across from one another so that visual contact was not obstructed at any time. The room was maintained on a 12-h light/dark cycle with no twilight, and temperature at 20-22°C with 50 f 10% relative humidity. 2.2. “P Spectroscopy The NMR system used for 31P data acquisition was a 4.7 Tesla (200 MHz) 33 cm horizontal bore superconducting magnet (Spectroscopy Imaging Systems, Sunnyvale, CA). A two-turn solenoidal radio frequency (RF) coil, 6 cm in diameter, was used for acquiring phosphorous (3’P) spectra, each of which contained 50 acquisitions. There was an active shield gradient set which can produce a maximum of 20 G/cm gradient field with a rise time of less than 290 ~.rsfrom O-98% gradient. The NMR system was controlled by a Sun SPARC II workstation (Sun Microsystems, Sunnyvale, CA). For each animal, the 31P RF coil was placed immediately above the skull after which a period of 5-10 min was allowed for coil tuning stabilization and for adjustment of the magnetic field

100 (199s)

129-137

131

homogeneity. Ten 31P NMR spectra were recorded first (spectral width = 3000 Hz) as control baseline. A non-selective RF pulse was used to excite the nuclei. For each pig, the RF power was determined prior to the baseline study to provide the maximum signal. The repetition time used was 1.2 s. All the spectra were treated with a 2 Hz line broadening before DC correction and baseline correction. The characteristic hump of 3’P spectrum was removed after baseline correction. Each spectrum was deconvoluted to obtain the peak area. Both the peak height and peak area were in the absolute machine intensity in which the scale was kept constant from spectrum to spectrum. The peak did not have a clear single peak due to the low line broadening (such as Pi peak in Fig. la). The whole area was used as the peak intensity. 2.3. Experimental design and procedures Anesthesia was provided by inhalation of isoflurane, 3-4% for induction and 1.5-2% for maintenance. In each experiment, the anesthetized animal was placed in a plastic cradle and positioned in the core of the NMR magnet. All pigs were placed on a Hamilton K-20 Aquamaticm rubberized heating blanket so that the body temperature was maintained at - 39.2”C. After the 10 baseline (control) spectra were recorded for each animal, NaCN (4.2 mg/kg), or H-7 (1 mg/kg) + NaCN (4.2 mg/kg), was administered i.v. H-7 was administered 30 min prior to NaCN as a pretreatment. Subsequently, post-NaCN levels of the metabolites were determined every minute from time zero (NaCN injected) to 60 min or until the animal died, as reflected by a domination of the 31PNMR spectra by Pi with a near-complete disappearance of PCr peak. In each case, animals that survived 60 min after receiving 4.2 mg/kg NaCN were considered to have survived the NaCN challenge and euthanatized by pentobarbital(lO0 mg/kg, i.v.) injection. To examine the effects of H-7 in the absence of cyanide, 31P-NMR spectral data were recorded at 1-min intervals for the 30-min pretreatment period immediately preceding NaCN injection. 2.4. Estimation of intracellular pH (pHi) The chemical shift difference (6) between Pi and

E. U. Maduh et al. / Toxicology 100 (1995) 129-137

132

PCr peaks was used to calculate pHi according to the formula pHi = 6.75 + lOgis(6 - 3.2715.69 - 6) a standard technique for estimating tissue pH in vivo, from 3’P-NMR spectroscopic data (Bore et al., 1982; Kimura et al., 1994).

2.5. Sources of drugs Isoflurane was a product of Anaquest (Madison, WI); pentobarbital sodium was obtained from AJ Buck (Timonium, MD). The PKC inhibitor H-7 dihydrochloride was purchased from Biomol (Plymouth Meeting, PA) and dissolved in 0.9% sodium chloride for injection, USP (Kendall McGaw, Irvine, CA).

Peak I.D. I =PME 2 = Pi 3=PDE 4 = PCI5 = y-ATP 6 = P-ATP 7 = wATP c

20 min

I5 nlin I 0 111 in 5 rnin Baseline

IO Illill 20

Peak 1.1). I =

r)r

lllill

IO nlin

I’MI:

I

win

H-7 PI-c-CN

Fig. 1. (a) “P-NMR spectra for pig no. 86-I injected with 4.2 mg@ NaCN alone. (b) 3’P-NMR spectra for pig no. 89-S pretreated with 1.0 mg/kg H-7, 30 min before 4.2 mgIkg NaCN was injected. a-ATP refers to the a-phosphate in ATP and the signal it generates.

E. U. Maduh et al. /Toxicology

Statistical analysis Statistical inferences were based on results of repeated measure analysis of variance with the Newman-Kuels post-hoc comparisons (Zar, 1984). Differences were accepted as signiticant if P s 0.05.

2.6.

3. Results In all experiments, baseline and post-NaCN spectra were acquired every minute through the course of each experiment. To simplify presentation, data were summarized by addition of the spectral information in 5-min intervals. Displayed in Fig. la, b are sample in vivo 31PNMR spectra representative of the two treatment groups before, and through 30 min after NaCN, 4.2 m&kg (Fig. la), and pretreatment with H-7 (1 mg/kg), followed by NaCN administration, i.v. (Fig. lb). As clearly evident in Fig. la, Pi peaks tended to increase with time after NaCN (4.2 mg/kg) was injected, whereas PCr peaks declined. The group receiving NaCN alone also exhibited a marked downward shift of the “P spectra (Fig. la), whereas, this effect does not occur if H-7 was administered before NaCN (Fig. 1b). Domination of the 3’P magnetic resonance spectra by Pi peak and the downward spectral shift are consistent with NaCN-induced histotoxic hypoxia, a char-

100 (199s)

129-137

133

acteristic of cytochrome oxidase (aa3) inhibition and loss of metabolic energy (Keilin, 1929; Schubert and Brill, 1968; Maduh et al., 1991). Therefore, bioenergetic parameters were obtained by analyzing ratios of the high energy phosphate metabolites relative to deconvoluted Pi peak area. There was marked decline in the PCr/Pi ratios which exhibited significant time-dependency, P < 0.05. Fig. 2 shows that with the 4.2 mg/kg dose given alone, the PCr/Pi ratio had fallen to 62% of its pretreatment mean value by 5 min after NaCN alone was given. By the 30-min time point, the PCr/Pi ratio had decreased to less than 33% of the pretreatment baseline. In comparison, with H-7 pretreatment, post-NaCN PCr/Pi ratio was 77% by the 5-min time point, and 52% of pre-NaCN value at the 30-min mark. Thus, pretreatment with H-7 blunted the decline in PCr/Pi ratios produced by NaCN, P < 0.05 (Fig. 2). Calculations of PCr/ATP ratios reveal timedependent reductions following NaCN injection and attenuation of these effects by H-7 (Fig. 3). At all times recorded, the mean ratios attained in the H-7-protected group were consistently greater than those seen in the unprotected (NaCN alone) group, P < 0.05. These ratios are consistent with the data shown in Fig. 1, and in the two groups examined, 215 of the unprotected and 415 of the H7-protected animals survived the NaCN treatment.

S.0 -1.5

o

1.0

l

0.0

1

= NaCN = NKN + H-7

-

0

0

5

IO

IS

1

20

3

30

Time (min) Fig. 2. Time-course of changes in PCr/Pi ratio before zero time point and after NaCN (4.2 mgkg, or preceded by H-7, I mg/kg) injected. Each point represents mean & S.E.M., n = 5.

was

E. II. Maduh et al. /Toxicology

134

100 (1995) 129-137

1.00 o = NaCN

E

2

= NaCN + H-7

l

/

o.75 -I 0.50 -

\

2

l\f \

0.25

-

0.00

L0

P

f

f

P-------

P

I

5

IO

I5

20

2.5

30

Time (min)

Fig. 3. PWATP ratio before zero time point and after the indicated NaCN dose or H-7 pretreatment (1 mg/kg) was injected. Each point represents mean l S.E.M., n = 5. (ATP = total 3’P peak intensities of y-, (Y-and &ATP).

We determined intracellular pH (pHJ at time points prior to, and after, NaCN in the presence and absence of H-7. Results of the pHi analysis before (time zero), post-NaCN, and H-7 + NaCN are displayed in Fig. 4. The pHi in the NaCN group consistently declined with time showing that intracellular acidification occurred. H-7 protected against cellular acidosis induced by NaCN. The mean values of pHi in those animals pretreated

with the PKC inhibitor were, at all times measurements were taken, higher than those obtained in the NaCN alone group with a statistical significance (P < 0.05) attained at the earlier 5-10 min time points, which corresponded with the earlier and most critical period in cyanide intoxication (Ballantyne, 1987). 3’P resonance spe ctra in the PME and PDE regions were also recorded and used as a means of

7.25 -

7.00 -

6.75 za

o l

= NaCN = NaCN + H-7

6.50 -

6.00 1

0

’ 0

5

IO

15 Time (min)

20

25

J 30

Fig, 4. Alterations in intracellular pH @Hi) before zero time point and after NaCN (4.2 mg/kg, or preceded by H-7, 1 mg/kg) was i!ija~%L &&I point represents mean f S.E.M., n = 5. lImtkate significant (P < 0.05) difference between the two groups.

E. V. Maduh et al. /Toxicology 100 (1995) 129-137

.

= PDE lor N,I(‘\ I_

+ H-7 -

IS

20

IO Time

135

1 I 25

.30

(min)

Fig. 5. Time-course of changes in peak intensities after NaCN (4.2 mg/kg, or preceded by H-7, I mg/kg) was injected at time zero. H-7 was injected 30 min before NaCN. Each point represents mean * S.E.M., n = 5.

assessing membrane phospholipid dynamics during NaCN toxicity. This analysis revealed few alterations in the 31P spectra for PME and PDE between the two treatment groups during the pretreatment phase of the experiment (Fig. 5). The H-7 and control values for PME and PDE were not significantly different from each other at the baseline time. 4. Discussion During cyanide toxicity, the synthesis of ATP is blocked with a concomitant accumulation of the lower energy phosphorous metabolites. In various model systems, cyanide toxicity provides a useful means for studying chemical hypoxia (Ahlemeyer and Krieglstein, 1989; Huang and Gibson, 1989). PKC inhibitors block cytotoxic injury and tissue death caused by experimental &hernia and hypoxia (Madden et al., 1991; Louis et al., 1993; Sahai et al., 1994), conditions which, like cyanide toxicity, stimulate reductions in cell energy phosphates as critical biochemical markers (Schanne et al., 1993). The present data demonstrate that H-7, a potent inhibitor of PKC (Kawamoto and Hidaka, 1984; Nishizuka 1986; Saitoh et al., 1991), reduced cyanide-induced cellular energy depletions and lethality in miniature pigs. Without pretreatment,

215animals given 4.2 mg/kg of NaCN survived the challenge, but 4/5 of the animals in the group pretreated with H-7 30 min before cyanide exposure survived. Thus, pretreatment with H-7 increased the number of animals surviving NaCN exposure and protected against cell energy reductions associated with acute cyanide toxicity. The use of in vivo NMR techniques in these cyanide studies failed to detect statistically signilicant changes in phospholipids (PME and PDE) after pretreatment with H-7 or administration of NaCN. Although the changes were not statistically significant, they may be biologically important because small changes in membrane phospholipid content may result in changes in membrane fluidity. Alternatively, the biological effects were small and the sensitivity of in vivo NMR was insufficient to detect these changes, while in vitro techniques (Maduh et al., 1988) utilized were adequate to detect these changes. However, the effects may not occur under in vivo conditions. In summary, the data illustrate time-course phosphorous energydepleting effects of NaCN. Pretreatment of the animals with a PKC inhibitor partially prevented these events and increased the number of survivors of NaCN challenge. We suggest that pharmacologic inhibition of PKC may be a useful therapeutic strategy to counter cyanide

136

E. U. Maduh et al. / Toxicology IW f1995) 129-137

toxicity in vivo (Maduh et al., 1993; Maduh and Baskin, 1994). Acbwledgements Dr. Maduh is a recipient of the U.S. National Research Council Resident Research Associateship Award. The authors are grateful to LTC Stephen J. Janny for providing expertise with the setup and optimization of the anesthetic inhalation device used throughout the study and to Dr. Tannis A. Johnson for her excellent veterinary assistance. Mention of products or company names does not constitute an endorsement by the authors, or by the affiliate institutions. Parts of this work have appeared as an abstract, Maduh et al. (1994) FASEB J. 8, A647. Reference-s Ahlcmeyer. B. and Krieglstein, J. (1989) Testing drug effects against hypoxic damage of cultured neurons during longterm recovery. Life Sci. 45, 835-842. Ballantyne, B. (1987) Toxicology of cyanides. In: Ballantyne and Marts (Eds), Clinical and Experimental Toxicology of Cyanides, Wright, Bristol, UK, pp. 41-126. Baskin, S.I., Horowitz, A.M. and Nealley, E.W. (1992) The antidotal action of sodium nitrite and sodium thiosulfate against cyanide poisoning. J. Clin. Pharmacol. 32, 368-375. Bore, P.J., Chart, L., Gadian, D.G., Radda, G.K., Ross, B.D., Styles, P. and Taylor, D.J. (1982) Noninvasive pHi measurements of human tissue using “P-NMR. In: Nuccitelli and Deamer (Eds), Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions. Kroc Foundation Series, Vol. IS, AR Liss, Inc., New York, pp. 527-535. Chance, B. (1989) What are the goals of magnetic resonance research? NMR Biomed. 2, 179-187. Hidaka, H. and Kobayashi, R. (1992) Pharmacology of protein kinase inhibitors. Annu. Rev. Phannacol. Toxicol. 32, 377-397. Huang, H.M. and Gibson, G.E. (1989) Phosphatidylinositol metabolism during in vitro hypoxia. J. Neurochem. 52, 830-835. Horn, G.E. and Johnson, J.D. (1987) Sulphur donors in cyanide intoxication. In: Ballantyne and Marrs @is), Clinical and Experimental Toxicology? of Cyanides, Wright, Bristol, UK, pp, 413-426. Ivankovich, A.D., Bravetznan, B., Kanuru, R.P., Heyman, H.J. and Paulissian. R. (1980) Cyanide antidotes and methods of their administration in dogs: a comparative study. Anesthesiology 52, 210-216. Keilin, D. (1929) Cytochrome and respiratory enzymes. Proc. R. See. B 104. 206-252.

Kimura, H.. Itoh, S., Kawamura, Y., Nakatsugawa, S. and Ishii, Y. (1994) Metabolic alterations in implanted human tumors after combined radiation and hyperthermia therapy measured by in vivo )‘P-MRS. MR Imaging 12, 109-l 19. Kawamoto, S. and Hidaka, H. (1984) l-(5-Isoquinolinesulfonyl)-2-methylpipcrazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. B&hem. Biophys. Res. Commun. 125, 258-264. Kruszyna, R., Kruszyna, H. and Smith, R.P. (1982) Comparison of hydroxylamine, 4dimethylaminophenoI and nitrite protection against cyanide poisoning in mice. Arch. Toxicol. 49, I91-202. Louis, J.C., Magal, E., Gerdes, W. and Seifert, W. (1993) Survival-promoting and protein kinase C-regulating roles of basic FGF for hippocampal neurons exposed to phorbol ester, glutamate and ischaemia-like conditions. Eur. J. Neurosci. 5, 1610-1621. Madden, K.P., Clark, W.M.. Kochhar, A. and Zivin, J.A., (1991) Effect of protein kinase C modulation on outcome of experimental CNS ischemia. Brain Res. 547, 193-198. Maduh, E.U.. Johnson, J.D., Ardelt, B.K., Borowitz, J.L. and Horn, G.E. (1988) Cyanide-induced neurotoxicity: mechanisms of attenuation by chlorpromazine. Toxicol. Appl. Pharmacol. 96, 60-67. Maduh, E.U.. Borowitz. J.L. and Horn, G.E. (1991) Cyanideinduced alteration of the adenylate energy pool in a rat neurosecretory cell line. J. Appl. Toxicol. I I, 97-101. Maduh. E.U., Porter, D.W. and Baskin, S.I. (1993) Calcium antagonists: a role in the management of cyanide poisoning? Drug Saf. 9, 237-248. Maduh, E.U. and Baskin, S.I. (1994) Protein kinase C modulation of rhodanesecatalyzed conversion of cyanide to thiccyanate. Res. Commun. Mol. Pathol. Pharmacol. 86. 155-173. Nishizuka, Y. (1986) Studies and perspectives of protein kinase C. Science 233. 305-312. Nishizuka. Y. (1992) Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607-614. Sahai. A., Patel, M.S.. Zavosh, A.S. and Tannen, R.L. (1994) Chronic hypoxia impairs the differentiation of 3T3-LI libroblast in culture: role of sustained protein kinase C activation. J. Cell. Physiol. 160, 107-112. Saitoh, T.. Masliah, E., Jin, L-W., Cole, GM.. Wieloch, T. and Shapiro, I.P. (1991) Protein kinases and phosphorylation in neurologic disorders and cell death. Lab. Invest. 64, 5%-616. Scharf, B.A., Fricke, R.F. and Baskin, S.I. (1991) Comparison of methemoglobin fanners in protection against the toxic effects of cyanide. Gen. Pharmacol. 23, 19-25. Schanne, F.A.X., Gupta, R.K. and Stanton, P.K. (1993) 3rPNMR study of transient ischemia in rat hippocampal slices in vitro. B&him. Biophys. Acta 1158, 257-263. Schubert, J. and Brill. W.A. (1968) Antagonism of experimental cyanide toxicity in relation to the in vivo activity of cytochrome oxidase. J. Pharmacol. Exp. Therap. 162, 352-359. van Heijst, A.N.P., Douze, J.M.C., van Kestren, R.G., van

E. lJ. Maduh et al. /Toxicology

Bergen, J.E.A.M. and van Dijk, A. (1987) Therapeutic problems in cyanide poisoning. Clin. Toxicol. 25, 383-398. Vick, J., Sass, N. and Herman, E. (1990) Cyanide poisoning in miniature swine: treatment with Na nitrite, dimethylaminophenol (DMAP) or hydroxylamine HCl. FASEB J. 4, A1089. Way, J.L. (1984) Cyanide intoxication and its mechanism of antagonism. Annu. Rev. Pharmacol. Toxicol. 24, 451-481. Way, J.L., Sylvester, D., Morgan, R.L., Isom, G.E., Burrows, G.E., Tamulinas, B. and Way, J.L. (1984) Recent perspectives on the toxicodynamic basis of cyanide antagonism. Fundam. Appl. Toxicol. 4, S231-S239.

100 (1995) 129-137

137

Weiss, S. and Ellis, L.B. (1933) Influence of sodium nitrite on the cardiovascular system and on renal activity in health, in arterial hypertension and in renal disease. Arch. Int. Med. 52, 105-l 19. Wing, D.A., Patel, H.C. and Baskin, S.I. (1992) The effect of picrylsulfonic acid on in vitro conversion of cyanide to thiocyanate by 3-mercaptopyruvate sulfurtransferase and rhodanese. Toxicol. In Vitro 6, 597-603. Zar, J.H. (1984) Biostatistical Analysis, 2nd Ed., Prentice-Hall, Englewood Cliffs, N.J.