Cyclic AMP in environmental toxicology

TIPS-December,

100 AMP and OIJ~ ignorance of the role of other cyclic nucleoaides. the conseSquences of PDE inhibitlion in cell function remain to a large extent unknown at present. There is no doubt that potent and specific inhibitors would 5e useful tools in elucidating the role GI PDEs in the regulation of cyclic mJcleotide levels and in analysing their funaion. It is too early to judge whether the development of such COWpound may provide useful therapeutic agents. Rending list I. Amer.M.S.(197S)L1leSr1.

17. I021-10.18. 2. Chasin, M. and Harris. U. N. (1976) In: P. Greengard and G. A. Robison teds). Advontes III CyCrrr Iiucleof6de Rtwarch, Vol. 7, Rawn Press. New Sork, pp. 225-264. 3. Demny-Waeldele. F. and S~ocler. J. C. (19:71

Err. 1. Pharmacol. 46. b&bb. ii. Ilien. 6.. Slierlr, A.. Lugnier. C.. Stocl:ln, J. C. and Landry. Y. (19783 Lochem. &ophy.v. Res. Commun. 8X486-492 5. Ktec. C. O., Crouch. T. H. and Krinks. M. H. f 1979) Biochemiw;v 18.722-729. 6. Sroc:el. J. C. (1978) In: J. C. Stocle~ ted.). Advances in Pharmacology and Therapeutics, Vol. 3, Pngamon Press. Oxford and New York.

pp. 181-192. 7. Srcwler.J. C. (1980) In: AcfplirPs de Chimte Thhcrapeutique, 7e stric. StiU de Chimie Therapeutique. Paris tin press). 8. Sutherland. E. W. and Rail. T. W. (1958) J. BiobChem. 232. 107?-1091. 9. Weiss. B. and Levin. R. M. (1978) In: P. Greengard and G. A. Robison teds). Advances in C_vclicNurkaride Research, Vol. 9. Raven Press, New York, pp. 285-303. 10. Wells. J. N. and Hardman. J. G. (1977) Ir.: P. Cireengard and G. A. Robison teds), Advances m Cyclrc Nucleolide Research, Vol. 8, Raven Press. New York.pp. It9-143.

Cyclic AMP in enwiranmen toxicology I

Radhey IL. Singhal Bparrmennr of Pharmacology. Faculiy of Health Scdences.School o: Medmne, L:nrversW of 011owc. Orrara. Canada RI.5 949.

of all kinds (e.g. heavy metals, food addidves., pesticide residues) have become an intrgral part of our environment and are known to affect human beings at man! levels of their vital physiological processes. Environmental toxicology deals with mcidental IeKposure of biologic tissue, pan!icuIarly of man. to chemicals that are basi~cally contaminants of his environment, food or water. Toxicologists and phnrmacologists alike are engaged in the stady of the calrses. conditions, effects, limits of safety 01: such exposure to chnnicals as well as in elucidating the cellular mechanisms whirt subserve their overt toxic effects’. The discovery of cyclic AMP stimulated research in diverse biological fields anid has now led to the establishment of a ho?,t of functions for this cyclic nu&otide in neuronal mechanisms, growl:h, cell differentiation and carter. lrnusculzr contraction and cardiovascular function, glandular secretion and immune response. Over the last five to six years, evidence also has emerged to show that cyclic AMP metabolism is markedly ahered :In response to exposure to various environmental chemicals. Adult or neonatal exposure to DDT and related halogenated ,Cd%.” L.” ,, ‘~ri.‘Hrl-.,J*i P,< IV.4 Contaminmas

1979

hydrocarbon insectllcides?, as well as to heavy metals such as cadmium or lead’,. produces marked disturbances in carbohydrate metabolism and evidence suggests that changes in the cyclic AMP system are intimately invoBverl in tissue responses to these environmerr.al pollutants. In the field o.i’ heavy metal toxicity, cadmium (0.1-l &g; p.o.) and lead (20-90 ppm; p.c.) exposure for 45-56 days has been shou:n to result in hyperglycosuria, hypoinsulinemia glycemia, and to enhance markedly the potential of

hepatir: and renal tissues to form glucose precursors from non-carbohydrate (gluconeogenesis)‘J. In addition, there was glucose intolerance probably associated with decreased pancreatic secretory activity as reflected by lowered insulinogenic indices and inhibitjon of phentolamine-stimulated insulin release. Similarly, when animals were treated intramuscularly with organochlorine pesticides such as p.p’-DDT (5-25 mg/kg). ,s-chlordane (5-25 mg/kg), heptachlor (3-15 Img/kg) or endrin (0.5-2 mg/kg) in small daily doses over a prolonged period (X-45 days), significant increases were seen in the quartet of hepatic and kidney cortex key gluconeogenic enzymes as well as in

Jean-Claude Stocet was born in I934 He took first and higher degree5 at rhe Universily of Paris. After restwrch m tionc* and the U.S.A., he was appointed professor qf phsrmacoloj~ ‘01 the Louis Pasteur University elf Strasbourg Faculry of Pharmav in 1968. His main mreresrs lie in rhe field of cyclic nucleorides ami calcium in vascular smooth muscle.

blood ancl urinary glucose and serum urea and a depression of liver glycogen stores’. It is noteworthy that enhancement of tissue cyclic AMP levels either by pharmacological means or following its exogenous administration can also result in several of the above noted biochemical effects. In this article, 1 shall confine my remarks IO a brief discussion of current evidence identifying the role of cyclic AMP as #an intracellular mediator of the actions of these two classes of environmental pollutants. Similarity in tissue responses to exogenous cyclic AhlIP and DDT, cadmialm and lead As set. out by Sutherland and his colleagues, before cyclic AMP can be implicated in the action of a given substance, the ability of exogenously administered cyclic AMP to mimic the effects of the chemical agent in question should be established. Administration of cyclic ,4MP (100 mg/kg) in two divided doses produced a significant rise in the activitbes of hepatic and renal .:ortex pyruvate carboxylase. phosphoenolpyruvate carboxykinase, fructose 1,6diphosphatase and glucose 6-phosphatase, elevated blood glucose and urea as well as lowered liver glycogen content, effects that were similar to those seen followiing exposure to P,P’-DDT. cr-chlordane, endrin, heptachlor, cadmium or lead. These results suggest that exogenous cyclic AMP is capable of mimicking certain effects of DDT and heavy metals on renal and hepatic carbohydrate metabolism’. It is interesting that

TIPS - L&ember.

1979

both actinomycnn D (40 &IO0 g; i.p.) and cyclohcximide (70 ~g/iOO g; i.p.), compounds that effectively prevented the cyclic AMP-stimulated rise in phoscarboxykinase phoenolpyruvate and glucose 6-phosphatase also were capable the p,p’-DDT-induced of blocking increases in vsrious key rate-limiting enzymes involved in the process of gluconeogenesis. Influence of DDT and heavy metal exposure on adenylate cyclase-protein base system

The ability of p,p -DDT to affect hepatic and renal adenylate cyclase activity has been examined both in vitro and in vivo2. Addition of 10” M p,p’-DDT to the incubation mixture failed to alter the activity of this cyclic AMP-synthesizing enzyme in both hepatic and kidney cortex homogenates. Whereas IO-! M concentration of the insecticide produced a significant increase in renal enzyme, maximal enhancement of liver and kidney adenylate cyclase was seen in the presence of IO” M p,p’-DDT In vivo treatment of rats with p,p’-DLIT (25 mg/kg; i.m.) produced a significant rise in hepatic and renal cyclic AMP levels as well as in the activity of both basal and fluoride-stimulated forms of adenylate cyclase. In contrast, p,p’-DDT did not seem to affect much the activity of the cyclic AMP-degrading enzyme, phosphodiesterase*. In rabbits poisoned with an organophosphate insecticide. Soman, a marked rise was noted in the concentration of plasma cyclic AMP by Stitcher et op. Similarly, Ihe activity of both basaland fluoride-stimulated forms of renal and hepatic adenylate cyclase as well as the endogenous levels of cyclic AMP have been shown to be elevated in animals treated with other cyclodiene insecticides, lead or cadmium. Administration of zinc (2 mg kg-’ day-‘) or selenium (I mg kg-’ day-‘), at the s,ame time as cadmium (1 mg kg-’ day-‘), effectively prevented the changes in hepatic cyclic AMP levels and ahe stimulatioln of basal, adrenaline as well as glucagcn-stimulated adenylate cyclase activity3J. It is of interest that hepatic adenylatr cyclase activity is also sensitive to the in virro action of an acaricide, Pliclran*. II is believed that cyclic AMP acts within the cell through the activation, of protein kinase(s) which phosphorylates various proteins such as histones, ribosomes and enzymes having key roles in responses. certain hormone-mediated

101

Following exposure to DDT, cyclic AMPdependent protein kinuse activity and cyclic AMP binding capacity were both significantly depressed in the hepatic cylosol fraction. Whereas the rndependent form of soluble cyclic AMP-protein kinase remained unaltered, the ratio of independent to dependent protein k;nase increased approximately two-fold. In contrast, phosphorylation of endogenous nuclear proteins, both in the presence and absence of cyclic AMP, was significanrly enhanced by pesticide treatment. As in the case of soluble binding capacity, there was a significant decrease in the ability ot cyclic AMP to bind to endogenous pro:rin in the nuclear fracGon_. Alterations in testicular and prostatic cyclic AMP metabolism following chronic cadmium treatment and subsequent withdrawal have also been investigated, and it was reported that changes in the adenylate cyclase and protein kinase system persisted even four weeks following the termination of daily exposure to this heavy metala. Furthermore, the increase in hepatic cyclic AMP levels seen with cadmium was associated with a decrease in the cyclic AMP-binding capacity of protein kinase. The kinase activity ratio, an indication of the relative activity of cyclic AMP-dependent- and -independent protein kinase, was reduced significantly in cadmium-treated animals with hepatic and renal protein kinases(s) being markedly altered following chronic exposure to this heavy metal’.

that

Soman

not

of cyclic

potentiation

the

A further evidence for the effect of chemicals on cyclic AMP metabolir,m was obtained by (a) studying cyclic [‘HIAMP formation in renal and

environmental

hepat? treated

slices obtained from pesticideanimals and incubating uith

(‘HI-adenosine slices from then

and (b) by obtaining tisr;uc normal

incubating

concentrations M) ,n vitro of labelled from

untreated them

rats and

with

\arl;ing

of the pesticide (IO’-IO-’

for 30 min. The enhancement cyclic nucleotide

tritiated

adenosine

produsrion

was time-

and

dcse-dependent

and significant changes in

cyclic [‘H]A%lP

lebels could be detecrrd

exposed to in animals p.p -DDT. c-chlordane, heprashlor or endrin. 4 significant increase in the biotransformation

of [‘Hladenosine

wz,

noted in slice5 excised from

into

c::slic A\lP rat? at

0 9, 1 and 3 h after pesticide treatment aith maximum stimulation beins seen a% esrly

as

30

min.

Furthermore,

Lbhrn

hepatic and renal cortices ohrained from normal rats acre incubated ttith \ar)inp amounts of DDT in ~1~0 :or 30 iTin. ma.ximum elevation in
\\as seen with

IO’

v

inre
although a statistically +n:ii
ri\c ua\ ot !JUC

_ ‘.

of

cyclic AMP does mediate the observed effects of various environmental chemicals on glucose homeostasis, treatment with a phosphodiesterase inhibitor such as theophylline would be expected to enhance the effects exerted by a submaximal dose of the polluant. N’hereas theophylline (10 mg/ 100 g; i.p.) alone produced small. but significant increases in gluconeogenic enzymes. ioncurrent Mid theophylline with treatment p,p’-DDT further augmented the asribiries of these hepatic and renal corre\ enzymes’. Simultaneous treatment with p,p’-DDT and theophylline also porenriated the insecticide-induced changes in blood glucose, serum urea and hspatic glycogen. As expected. treatment of DDT-treated animals with caffeine augmented the action of this pesticide on cyclic [‘HIAMP formation in kidney cortex slices. Stitcher ef a/.” also reported if

incrcared 4MP

Pesticide influence on cyclic [‘HIAMP synthesis from tritiated adenosine

conientrations’

Methyl xanthine-indwed insecticide action

only

in rabbit p1asrr.a. but that theophylline potenriatrd the toxicity of this compound. concentration

Pharmacological modulation [‘HIAMP formation

of cyclic

The influence of sebera! agent\ knoun fo affect carbohydrate and $y
tritiated

adcnosinr

has also been

studied.

In contrast to methyl xanrhine<. of phosphodlestera stimulation

imidazole

(-10 me IOU p)

slgnifi
louered the peqti
intraperironeal

either

h~drarinc

admimrtrailan

(26

mg ILK, p) Gr

propranolol (‘0.3 mg IN 01 to rats gibcn DDT (10 mg 100 g) resulted m a loucrinp of rhe pesticide-stimulated

enhanzemenr

in renal cyclic [‘HjAhlP. prostaglandin Ei (10 qg, 100 g) by itself, failed to exert any

significant

kidney

effect.

slices were

L.ikewise,

incubated

eously Hith p,p’-DDT glandin E! or F?, (IO”

\%hen

simultan-

and either prosta%I). the insrcticide-

TIPS - December,

IK?

stimulated rise in cyclic nucleotide formation remlained unaffected. However, concurrent incubdtion (in virfol of renal slices with DDT {IO* w) and either hydrazine (IO-’ hi). imidazole (IO mM) or peopranolol (lO+ ,a) significantly lowered the amoum of cyclic I’HIAMP formed when compared with the values seen with pesticide alone. These ;results suggest that cyclic [‘HIAMP production from tritiated adenosine is not only markedly affected by cyclodiene insecticifdes, but is subject to further modulation iby several pharmacological agentsz.7.‘“. Enriroamental snd celioirr

toxicology-cyclic

AMP

less, in conjunction with other tests, monitoring of urinary and/or circulatory cyclic AMP levels may represent a noninvasive index of exposure to certain P nvironmental substances. Acknowledgements

The author acknowledges with gratitude the valuable contributions of Drs S. Kacew and Z. Merali and Miss A. Stevenson. This research was supported by grants from the Medical Research Council and Health and Welfare Canada.

W. and tinrry. V. I..

Mcmli. Z.. liacew, S.. tioch. ft. B. and Singhal. R.L. (1975)Gm. Phorn~oco/. 6.299-301. 10. Yaccw. S. and Sinyhal. R. I.. (19741 B1w4em. J. 142. 145-152. II. Kaccw. S.. Mcrali. Z. iandSinghal. R. 1.. (1976) Toxicol. Appl. Pharmacol. 38,145- 156. 12. Weiss. 8. and Greenberg. L. H. (1975) In: C’yrlic’ Nwleorides in Disease, B. Weiss (cd.). University Park Press. Baltimore, MD. pp. 269-320. 13. Coulson. S. and Bowman, R. H. (1974) ri_fr sn’. 14.545-556.

Reading lit

I. 2.

growth

In addition to the effects of environmental chemicals on f.he body’s glucose homeostasis. changes In cellular growth have been observed foilowing prolonged exposure IO heavy melals. It was shown that in the lung, cadmium administration increased the incorporation of thymidine into DNA which was preceded and accompanied by a rise in pulmonary cyclic AMP concentration”.. Likewise, the depression in kidney DNA synthesis was associated with a reduction in cyclic nucleotide content. Neonatal exposure to lead also enhanced the incorporation of thymidine into renal, hepatic and pulmonary DNA with the observed response being associa.ted with a rise in tissue cyclic AMP. thus supporting the concept that cyclic A&VIPmodulation may be an essential prerequisite for triggering cellular DNA synthesis. At the present time, it is not known whether monitoring plasma cyclic AMP levels or its urinary excretion in human subjects exposed to, or only suspected of being exposed to certain environmental contaminants, can he used as a biochemical index for measuring their toxicological potential on body metabolism. Although controver’,y continues to exist. some investigators h.we found differences in the excretion of cyclic AMP in patients with various types of psychiatric illnesses undergoing treatmerit with psychopharmacological agents such as lithiumLz. The rena: handling of cyclic AMP is complex, ill-defined. and seems to involve glomerular filteration, tubular secretion and probably tubular reabsorption”. Until such time that we clearly understand the kinetics of extracellular cyclic AMP. any data Ott circulating cyclic ,lMP or its urinary excretion following exposure to hmdouc chemicals in our environment, must be viewed with caution, Nonethe‘,E’s.6 -.ir:r H.-a.#$&‘m‘ti.;r~P,ct, ,974

8. Slltchcr. D. L.. Hnrris, I y, ( 1975) Fed. Proc. 34. 737.

1979

3. 4.

5. 6. 7.

Singhal, R. L. and Thomas, J. A. (co-cds) (19791 In: Monograph on Lead Foxicqv. Urban & Schwarzmbcrg. Baltimore. MD (in press). Kacew. S. and Singhal. R. L. (1974) J. Pharmacol. Erp. Fher. 188.265-276. Merali. 2. and Singhal, R. L. (1975) J. Pharmacol. &xp. Fher. 195.58-S. Singhal. R. L. and Merali. Z. (1979) In: J G. Mennear @.I.). Cadmium Foxici~.v. Madern Dekkcr Pharmacology -Fo.rwolo~v. Marcel inc.. New York. pp. 61-112. Swenson. A.. Merali, Z.. Kaceu. S. and Singhal. R. L. (1976) Foxicofonf 6.265-275, Hrdina. P. D.. Singhal. R. I.. anld Ling. G. M. (1975) Adv Phormard. Chemorher. 12,31-84. Singhal. R. L. and Kaccw. S. (1976) Fed. Proc. 35,2618-2623.

The author is Professor and Chairman in the Department OJ Pharmocolo#y. Faculfy of Health Sciences, School of Medici,ne. University 01 Qrtawa. Prior ro his move, Dr Sinkhal worked wirh Professor George Weber at Indiana Universify, School of Medicine, Jrom 1962-1966.

Kinetic events deteminina the effects of cardiac glycosides Heizlz LUllmann, Thies Peters and Albrecht Ziegler Deparcmem of P~rmaco&?.v.

Universiry of Kiel. Hospitalswaw

Drugs pose kinetic problems with drug dir;position), the transformation

on three different

4-6, D-2XHl Kiel. F.R.G.

levels: (1) pharmacokinetics

(2) receptor kinetics, and (3) tranformation

of the drug-receptor

interaction

(dealing

kirtetics (describing

into effects). The events occurring

on these three levels have a highly complex interdependence.

Receptor and transforma-

tion kinetics of rutturally occurn’ng cardiac glycosides congenecr deserve more attention in view of t,heir therapeutic

and their semisynthetic importance.

Kinetic events on lbree different

levr-Is

mentioned above, the time cout?,e of drug effects, can in principle, be determined by kinetic events on three different levels. This is schematically presented in Fig. 1. In the first place. most drugs have to be transported to their sites of action at the plasma membrane or at intracellular structures. Thii proecess of time-dependent changes of drug concentrations in the so-called biophase is the subject of pharmacokinetics, at least as far as the As

extracellular concentrations are concerned. Subsequently, on a second level of kinetics the time course of the drug interaction with the specific binding sites (receptors) or unspecific binding sites has to be taken into account; receptor kinetics. Finally, on a third level, the time course of the transformation of the receptor occupation into the biological effect has to be considered; tranqformation kinet& The time course of an effect can be determined by events on either level. The