TOXICOLOGY
AND
APPLIED
Cadmium
PHARMACOLOGY
M,143-150
(1975)
and Vascular
Reactivity
in the Rat’
MICHAEL C. PORTER, TOM S. MIYA, AND WILLIAM F. BOUSQUET Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 Reeeived June 3,1974; accepted May 21, 1975
Cadmium and Vascular Reactivity in the Rat. PORTER, M. C., MIYA, T. S., AND BOUSQUET, W. F. (1975).Toxicol. Appl. Pharmacol.34,143-150. Cadmiumhas beenreported to induce hypertensionaccompaniedby decreasedvascular responsiveness to autonomic drugs in the rat and rabbit. Cadmium acetateadministrationto femaleSprague-Dawleyrats failed to elevate blood pressure, although cadmium-treated animals exhibited decreasedblood pressureresponsesto intravenously administerednorepinephrine, acetylcholine,isoproterenol, and atropine. Aortic strips from cadmium acetate-treatedanimals demonstrateddiminishedreactivity to angiotensin, epinephrine, barium, and tyramine. It is concluded that cadmium treatment of the rat producesdesensitizationof the vasculature to vasoconstrictor and vasodilator substancesindependentlyof its ability to produce hypertension and, thus, that these facets of its biological activity are unrelated.
A number of toxic manifestations
including growth retardation (Wilson et al., 1941) anemia (Fitzhugh and Meiller, 1941),proteinuria (Friberg, 1955),carcinogenesis(Gunn et al., 1964) testicular necrosis (Parizek and Zahor, 1956), central nervous system lesions (Gabbiani et al., 1967a), renal damage (Axelsson and Piscator, 1966), and hypertension (Schroeder et al., 1966; Thind et al., 1970) have been reported following oral or parenteral administration of cadmium (Cd). Although several organ systems are involved in the expression of Cd toxicity, it is clear that the vascular actions of the metal may underlie such organ-specific damage. The testicular necrosis produced by Cd is the product of damage to the testicular blood vessels (Gunn et al., 1963) as is the ability of the metal to damage sensory ganglia (Gabbiani et al., 1967b). Placental and ovarian necrosis produced by Cd in rodents may also relate to a fundamental vascular insult (Chiquoine, 1965; Kar et al., 1959). Schroeder et al. (1970) examined the effects of vasoactive substances on the blood pressure of Cd-treated, male Long-Evans rats. Those animals which had received Cd (10 ppm of Cd in drinking water for 2-4 mo) had higher blood pressures than control animals, but exhibited depressed responsiveness to the vasopressor effects of norepinephrine and angiotensin. From these findings, the authors concluded that Cd hypertension may be accompanied by diminished vascular reactivity. Thind et al. (1969) studied the responsiveness of the thoracic aorta from young New Zealand White male rabbits made hypertensive by six to eight weekly injections of Cd. They found the contractile ’ This work wassupportedby Public Health ServiceEnvironmentalToxicology TrainingGrant 2TOl ESOOO71-06. Appreciationis expressed to CibaLaboratoriesfor providing angiotensinamide. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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response to angiotensin to be significantly decreased in aortic strips from the hypertensive animals. In recent studies on Cd-hypertensive dogs and rabbits, Thind (1974) has observed a decreased responsiveness of the isolated carotid artery from Cd-hypertensive dogs to angiotensin, norepinephrine, and serotonin. Similar changes were observed with angiotensin acting on the isolated rabbit aorta; the response of this preparation to norepinephrine, however, was unaltered from that of normotensive controls. Measurement of the wall thickness of the vessels used in these in vitro studies revealed a similar species difference. Rabbit aortic wall thickness was unchanged whereas in the dog carotid artery this value was increased by the Cd treatment. These observations leave unanswered the question of whether a causal relationship exists between Cd-induced hypertension and changes in vascular reactivity. In the present study, the administration to male and female Sprague-Dawley-derived and female hooded Long-Evans rats of the dosage schedule of Cd acetate employed by Schroeder et al. (1966) produced no increase in blood pressure in any of the Cd-treated animals as reported by Porter et al. (1974). It was decided to conduct studies on the vascular reactivity of these Cd-treated rats, to determine if altered vascular responsiveness to vasoactive substances is dependent upon the existence of the hypertensive state following Cd administration, as indicated by the reports of others referenced above. METHODS
Animals. Female Sprague-Dawley-derived rats (Laboratory Supply Co., Indianapolis), 91-94 days of age, were employed in this study. Animals were housed in stainless steel community cages in temperature (22-24°C) and light (14-10 light-dark) controlled rooms and allowed free access to laboratory chow (Allied Mills, Chicago) and distilled water. Indirect blood pressure measurements were made on lightly ether-anesthetized rats by the tail-cuff method of Friedman and Freed (1949). Preliminary experiments showed no significant difference between blood pressures recorded while rats were under light ether anesthesia as compared to animals under pentobarbital anesthesia. Animals were allowed 5 days to acclimate to the laboratory environment and diet, and two control blood pressure recordings at 2-day intervals were taken on all animals prior to Cd administration. Arterial blood pressure responseof the Cd-treated rat to various autonomic agonists andantagonists. A control group of 12 female, 91-day-old Sprague-Dawley-derived rats
received saline (2.0 ml/kg, ip) while a similar group of 12 animals received Cd (2.0 mg Cd/kg, ip) as the acetate. On day 21 following this initial injection, a second dose of Cd (1.0 mg Cd/kg, ip) or saline (1.0 ml/kg, ip) was administered. From days 14-27, following the second administration of Cd, these animals were employed for determination of blood pressure responses to various autonomic drugs. The drugs and dose levels administered included: epinephrine HCl (0.5 pg/kg in 0.01 N HCl); norepinephrine bitartrate (0.5 pg/kg in 0.01 N HCl); acetylcholine iodide (0.1 pg/kg in 0.01 N HCI); isoproterenol HCI (0.01 ,ug/kg in 0.01 N HCI); atropine sulfate (0.5 mg/kg in 0.9 % NaCl solution); and propranolol HCI (1 .Omg/kg in 0.9 % NaCl solution). Animals were anesthetized with DialR-urethane solution (0.6-0.8 ml/kg, ip). Each animal was then secured on a rat board, a tracheal cannula inserted, the left femoral vein
CADMIUM
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14.5
cannulated (PE 20 tubing) for intravenous administration of drugs, and the right carotid artery cannulated (PE 90 tubing) for direct, continuous blood pressure recording employing a P-1000-A pressure transducer and a Physiograph. The carotid cannula contained sodium heparin (100 USP units/ml.) Drugs were injected via the femoral vein in volumes of 0.07-0.09 ml and washed in with 0.10 ml of 0.9 % NaCl solution. Initial systolic blood pressures were between 110 and 150 mmHg. In a few animals where the systolic pressure fell below 90 mmHg during administration of the drug sequence, 5 % bovine albumin was infused until the pressure was restored to greater than 90 mmHg. The sequence of drug administrations was as follows: I. epinephrine; 2. norepinephrine; 3. acetylcholine; 4. isoproterenol (drugs l-4 were then repeated in the same sequence); 5. atropine; 6. acetylcholine; 7. propranolol; 8. isoproterenol; 9. epinephrine; 10. norepinephrine. The control administration of 0.9 % NaCl solution or 0.01 N HCl(0.2 ml) caused insignificant changes in blood pressure. Following the injection of each drug, the maximum rise or fall in blood pressure was recorded. The next injection was not made until the blood pressure had returned to the value obtained before the drug administration and had stabilized. For each animal the blood pressure change following epinephrine, norepinephrine, acetylcholine, and isoproterenol was taken as the average change for the first two injections of each of these drugs, prior to the administration of atropine and propran0101. The blood pressure change following atropine and propranolol was based on a single administration of these drugs to each animal. The mean maximum rise or fall in blood pressure caused by each drug was then compared for animals in the control and Cd-pretreated groups by means of Student’s t test. EfSect of Cd pretreatment of the rat upon the reactivity of aortic smooth muscle to various agonists. A control and a treatment group (16 rats each) of female 94-day-old,
Sprague-Dawley-derived rats received saline (2.0 ml/kg, ip) or Cd (2.0 mg Cd/kg, ip) as the acetate, respectively. On day 21 following this initial injection, a second dose of Cd (1.0 mg Cd/kg, ip) or saline (1.0 ml/kg, ip) was administered. From days 9-37 following the second administration of Cd, studies were conducted on aortic strips from control and Cd-treated animals to determine if the reactivity of this smooth muscle preparation was modified by Cd treatment. Dose-response relationships for the following agents were determined on each aortic strip: tyramine HCl (10-6-10-3 M); angiotensin amide (10-9-10-6 M); epinephrine HCl (10-9-10-5 M); and barium (10V6-IO-” M). All solutions remained stable throughout the period of experimentation, under the conditions of storage employed. Barium was kept at room temperature (22-24”C), while all other drugs were refrigerated (4--5°C). Angiotensin was adjusted to pH 2 with 0.01 N HCl to assure its stability (Thymann, 1968). Tyramine and epinephrine were also adjusted to pH 2. Barium solution was prepared from barium hydroxide which was then adjusted to pH 6 with 2.0 N and 0.01 N HCI. Cd-treated and control animals were ether anesthetized after which the descending thoracic aorta was rapidly excised and placed in cold, oxygenated Krebs-Henseleit solution (Krebs, 1950). The aorta was then cut along a loose spiral with microdissecting scissors by the method of Furchgott and Bhadrakom (1953). This spiral was then cut into two segments of 2 cm each which were mounted in a muscle bath containing 75 ml of Krebs-Henseleit solution. There was no significant difference in the reactivity of the
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upper and lower portions of the aortic strip as measured by the response to epinephrine. The contractile force of aortic strips was measured isometrically with a Grass Force-displacement Transducer FT03C and recorded on a Grass model 5 Polygraph. One gram of tension was placed on all strips and the strips allowed to equilibrate for 2 hr before beginning the drug sequence. After the strips had remained in the bath for 105 min, fresh bathing solution was added and the first dose-response relationship started 15 min later. All drug additions were cumulative with each progressive concentration being added at the peak of the previous response. Following each dose-response measurement, fresh bathing solution was added and the strips allowed to return to baseline tension over a 25-min period. At this time fresh solution was again added and the next dose-response measurement was started 5 min later. The bath temperature was maintained at 37.5”C and the pH at 7.3-7.5. A gas mixture of 95 % O2 and 5 % COz was bubbled through the bath continuously. A volume of 0.1 ml of drug solution was added at each concentration step of every dose sequence except at the 3 x 10m4M and 1 x 10m3 M concentrations of barium. In these instances it was necessary to add 1.0 ml of the barium solutions to the 75-ml muscle bath due to the limited solubility of barium. RESULTS Arterial blood pressure response of the Cd-treated rat to various autonomic agonists and antagonists. Weekly recordings of blood pressures (indirect) following Cd administration disclosed no significant differences between Cd-treated and control rats prior to the use of these animals in subsequent studies of blood pressure responses to various autonomic drugs (Porter et al., 1974). Data in Table 1 show the blood pressure response to various autonomic drugs elicited upon their administration to Cd-treated and TABLE 1 ARTERIAL
BLOOD PRESSURE RESPONSE OF THE CADMIUM-TREATED TO VARIOUS AUTONOMIC AGONISTS AND ANTAGONISTS“
RAT
Systolic blood pressure change (mmH8 +- SE) Drug and dose Epineprhine (0.5 fig/kg) Norepinephrine (0.5 Dug/kg) Acetylcholine (0.1 pg/kg) Isoproterenol(O.01 pg/kg) Atropine (0.5 mg/kg) Propranolol(1 .Omg/kg)
Control T21f 2 (8)b 131f 3 (8) $23+ 2 (8) $18f4(8)
Treated T21+ 1 (7) NS’ f22 + 1 (7)d $16+ 2 (7)d $13+ 3 (7) NS
$21 + 5 (8)
J12 + 2 (7) NS
$34It 5 (7)
$34+ 4 (6) NS
a Sprague-Dawley-derived rats received cadmium (2.0 mg Cd/kg and 1.0 mg Cd/kg, ip) as the acetate, or saline (2.0 ml/kg and 1.0 ml/kg, ip) 21 days apart. The blood pressure responses of control and cadmium-treated animals to intravenously administered autonomic drugs were determined starting 14 days after the second dose of cadmium. * Number of animals per group. c NS=Not significantly different (p > 0.05) relative to saline controls. d Significantly different (p < 0.05) relative to saline controls.
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control rats. A significant (p < 0.05) decrease in the vascular reactivity to norepinephrine and acetylcholine was seen in the Cd-treated animals. A substantial, though not significant, reduction in blood pressure response was also seen in the Cd-treated animals following isoproterenol and atropine. Responses to epinephrine and propran0101 did not differ for the control and Cd-treated groups. Effect of Cd pretreatment of the rat upon the reactivity of aortic smooth muscle to various agonists. Aortic strip dose-response curves for angiotensin, epinephrine.
lJJ .05t( I” w : 2
.04-
.03 Y F y .02-
E B
.Ol -
0 c 4
00 I
-.OJ 10-s
CONCENTRATION
OF
ANGIOTENSIN
I
I 10-a (moles I liter )
1. Effect of cadmium pretreatment of the rat on contractility of thoracic aortic strips to angiotensin. Female, Sprague-Dawley-derived rats received cadmium (2.0 kg Cd/kg and 1.0 mg Cd/kg, ip) as the acetate, or saline (2.0 ml/kg and 1.Oml/kg, ip) 21 days apart. The response to angiotensin of replicate aortic strips from 10 control and 10cadmium-treated animals was measured starting 9 days after the second dose of cadmium. Standard errors are represented by vertical bars. FIG.
IO-*
10-S CONCENTRATION
OF
10-T EPINEPHRINE
(rndos
10-s I l,,er )
lo-’
FIG. 2. Effect of cadmium pretreatment of the rat on contractility of thoracic aortic strips to epinephrine. See Fig. 1 for experimental conditions. The response to epinephrine of replicate acrtic strips from 10 control and 10 cadmium-treated animals was measured starting 9 days after the second dose of cadmium. Standard errors are represented by vertical bars.
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PORTER,MOYAANDBOUSQUET
CONCENTRATION
OF BARIUM
( molss/litrr)
FIG. 3. Effect of cadmiumpretreatmentof the rat on contractility of thoracic aortic stripsto barium.SeeFig. 1 for experimentalconditions.Theresponse to bariumof replicateaorticstripsfrom 10 control and 10cadmium-treated animalswasmeasured starting9 daysafter the seconddoseof cadmium.Standarderrorsarerepresented by verticalbars. .16cti +I 0
.14-
1
.lO-
.12-
.06 ?I
ii
.06-
? z 8
.04-
" .02F fj om
-ye
5
I P
Q I
I
10-s CONCENTRATION
OF TYRAMINE
lo-’ (moles /liter)
10-S
FIG. 4. Effect of cadmiumpretreatmentof the rat on contractility of thoracicaortic stripsto tyramine.SeeFig. 1 for experimentalconditions.Theresponse to tyramineof replicateaortic strips from 10control and 10cadmium-treated animalswasmeasured starting9 daysafter theseconddose of cadmium.Standarderrorsarerepresented by verticalbars.
barium, and tyramine are shown in Figs. 14. A general feature of these responsesis a tendency toward a shifting of each line to the right along the dose axis, and a lower maximum response of the aortic strips from the Cd-treated animals to the several agonists, when compared to strips from control rats. DISCUSSION
The intravenous administration of vasoactive substancesto Cd-treated and control rats demonstrated a significantly (p < 0.05) decreased vascular responsiveness, as
CADMIUM
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determined by the magnitude of the blood pressure response, to norepinephrine and acetylcholine and a substantial (28 and 42x), though not significant, decrease in vascular responsiveness following isoproterenol and atropine in the Cd-treated animals. No difference between control and treated animals was apparent following epinephrine or propranolol. Studies involving the direct addition of vasoactive substances to aortic strips. showed a decreased sensitivity of such strips from Cd-treated animals to angiotensin, epinephrine, barium, and to the indirect-acting agonist, tyramine. The possibility exists that many of the pathophysiological actions of Cd are related to the interaction of Cd with body stores of essential ions, especially calcium and zinc. Specificity of the toxic actions of Cd are difficult to assess in view of the fact that such toxicity may be inhibited by several other metals. For example, the testicular necrosis produced by Cd may be blocked by zinc (Parizek and Zahor, 19X$ selenium (Kar et aE., 1960), and manganese and cobalt (Gunn and Gould, 1970). Increased levels of calcium in drinking water have been reported to suppress Cd-induced hypertension in rats (Schroeder et al., 1967). Friberg et al. (1971) reported one of the major toxic signs of the Cd-induced “Itai-itai” disease to be weakening of the bone structure, which strongly suggests calcium depletion. It is well documented that calcium ion is the agent activating contractile elements in both skeletal and smooth muscle fibers (Hudgins and Weiss, 1968; Hurwitz et al., 1973). Hurwitz et al. (1973) reported that a specific intracellular pool of calcium supports the contractile response to a significant degree in the main pulmonary artery of the rabbit. If the same is true of the rat, depletion of such an intracellular pool of calcium by Cd might lead to a decreased contractile response of the vasculature. Regardless of the precise mechanism(s) involved, the present findings suggest that a decreased vascular reactivity to both vasoconstrictor and vasodilator substances follows Cd administration to the Sprague-Dawley-derived rat. It is a major finding of this study that the ability of Cd treatment of the rat to alter vascular sensitivity to vasoconstrictor and vasodilator agents was shown to be independent of the ability of the metal to induce hypertension. REFERENCES AXELSSON, B., AND PISCATOR, M. (1966). Renal damage after prolonged An experimental study. Arch. Environ. Hlth. 12, 360-373. CHIQUOINE, A. D. (1965). Effect of cadmium chloride on the pregnant
exposure albino
to cadmium.
mouse.
J. Reprod.
Fertil. 10,263-265. FITZHUGH,
0. G.,
AND MEILLER,
F. H. (1941). The chronic
toxicity
of cadmium.
J. Pharmacof.
Exp. Ther. 72, 15. FRIBERG, L. (1955). Iron and liver administration in chronic cadmium poisoning and studies of the distribution and excretion of cadmium. Acta Pharmacol.Toxicol. 11, 168-178. FRIBERG, L., PISCATOR, M., AND NORDBERG, G. (1971). Cadmiumin the Environment, pp. 111-128. CRS Press, Cleveland, Ohio. FREIDMAN, M., AND FREED, S. C. (1949). Microphonic manometer for indirect determination of systolic blood pressure in rat. Proc. Sot. Exp. Biol. Med. 70,67O-672. FURCHGOTT, R. F., AND BHADRAKOM, S. (1953). Reactions of strips of rabbit aorta to epinephrine, isopropyl arterenal, sodium nitrate and other drugs. J. Pharmacol.Exp. Ther. 108,
129-143. GABBIANI, sensory
G., GREGORY, A., AND BAIC, D. (1967a). Cadmium-induced ganglia. J. Neuropathol.Exp. Neural. 26,498-506.
selective
lesions
of
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G., BAIC, D., AND DEZIEL, C. (1967b). Studies on tolerance and ionic antagonism for cadmium or mercury. Can. J. Physiol. Pharmacol. 45,443-450. Gum, S. A., GOULD, T. C., AND ANDERSON, W. A. D. (1963). The selective injurious response of testicular and epididymal blood vessels to cadmium and its prevention by zinc. Amer. J. Pathol. 42,685-702. GUNN, S. A., GOULD, T. C., AND ANDERSON, W. A. D. (1964). Effect of zinc on cancerogenesis by cadmium. Proc. Sac. Exp. Biol. Med. 115,653-657. GUNN,S. A., AND GOULD, T. C. (1970).Cadmiumand other mineral elements.In The Testis A. D. Johnson,W. R. Gomer, and N. L. Van Demask,pp. 377-481.Academic Press,Inc., New York. HUDGINS, P. M., AND WEISS, G. B. (1968).Differential effectsof calciumremovalupon vascular smooth muscle contraction induced by norepinephrine, histamine and potassium.J. Pharmacol.Exp. Ther. 159,91-97. HURWITZ, L., FITZPATRICK, D. F., DEBBAS, G., AND LANDON, E. J. (1973). Localization of calciumpump activity in smoothmuscle.Science179, 384-386. KAR, A. B., DAS, R. P., AND KARKUN, J. N. (1959).Ovarian changesin prepubertalrats after treatment with cadmiumchloride. Acta Biol. Med. Ger. 3, 372-379. KAR, A. B., DAS, R. P., AND MUHERJJ, F. N. I. (1960).Prevention of cadmiuminducedchanges in the gonadsof rat by zinc and selenium-a study in antagonismbetweenmetalsin the biological system. Proc. Natl. Inst. Sci. India B26 (Suppl. 40), 40-50. KREBS, H. (1950).Body sizeand tissuerespiration. Biochem.Biophys.Acta 4,249-269. PARJZEK, J., AND ZAHOR, Z. (1956).Effect of cadmiumsaltson testiculartissue.Nature(London) GABBIANI,
177,1036-1037.
M. C., MIYA, T. S.,AND BOUSQUET, W. F. (1974).Cadmium:Inability to inducehypertensionin the rat. Toxicol. Appl. Pharmacol.27,692-695. SCHROEDER, H. A., KROLL, S. S., LITTLE, J. W., LIVINGSTON, P. O., AND MYERS, M. A. G. (1966).Hypertensionin rats from injection of cadmium.Arch. Environ. H&h. 13,788-789. SCHROEDER, H. A., NASON, A. P., AND BALASSA, J. J. (1967). Trace metals in rat tissues as influencedby calciumin water. J. Nutr. 93, 331-335. SCHROEDER, H. A., BAKER, J. T., HANSEN, N. M., JR., SIZE, J. G., AND WISE, R. A. (1970). Vascularreactivity of rats alteredby cadmiumand a zinc chelate.Arch. Environ. Health 21, PORTER,
609-614.
G. S., STEPHAN, K. R., AND BLAKEMORE, W. S. (1969). Vascular responsiveness in cadmium-inducedhypertension.Fed. Proc. 28,403ABS. THJND, G. S., DARREMAN, G., STEPHAN, K. R., AND BLAKEMORE, W. S. (1970). Vascular reactivity and mechanicalpropertiesof normal and cadmium-hypertensiverabbits. J. Lab. Clin. Med. 76, 560-568. THJND, G. S. (1974).Blood vesselwall characteristicsin experimentalhypertension.Angiology THJND,
25,752-763. M. (1968).The stability of angiotensinamide in aqueous solution. DanskTidsskrift Farmaci 42,225-234. WILSON, R. H., DEEDS, F., AND Cos, A. J., JR. (1941).Effects of continuedcadmiumfeeding. J. Pharmacol.Exp. Ther. 71,222-235. THYMANN,
AND
APPLIED
Cadmium
PHARMACOLOGY
M,143-150
(1975)
and Vascular
Reactivity
in the Rat’
MICHAEL C. PORTER, TOM S. MIYA, AND WILLIAM F. BOUSQUET Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 Reeeived June 3,1974; accepted May 21, 1975
Cadmium and Vascular Reactivity in the Rat. PORTER, M. C., MIYA, T. S., AND BOUSQUET, W. F. (1975).Toxicol. Appl. Pharmacol.34,143-150. Cadmiumhas beenreported to induce hypertensionaccompaniedby decreasedvascular responsiveness to autonomic drugs in the rat and rabbit. Cadmium acetateadministrationto femaleSprague-Dawleyrats failed to elevate blood pressure, although cadmium-treated animals exhibited decreasedblood pressureresponsesto intravenously administerednorepinephrine, acetylcholine,isoproterenol, and atropine. Aortic strips from cadmium acetate-treatedanimals demonstrateddiminishedreactivity to angiotensin, epinephrine, barium, and tyramine. It is concluded that cadmium treatment of the rat producesdesensitizationof the vasculature to vasoconstrictor and vasodilator substancesindependentlyof its ability to produce hypertension and, thus, that these facets of its biological activity are unrelated.
A number of toxic manifestations
including growth retardation (Wilson et al., 1941) anemia (Fitzhugh and Meiller, 1941),proteinuria (Friberg, 1955),carcinogenesis(Gunn et al., 1964) testicular necrosis (Parizek and Zahor, 1956), central nervous system lesions (Gabbiani et al., 1967a), renal damage (Axelsson and Piscator, 1966), and hypertension (Schroeder et al., 1966; Thind et al., 1970) have been reported following oral or parenteral administration of cadmium (Cd). Although several organ systems are involved in the expression of Cd toxicity, it is clear that the vascular actions of the metal may underlie such organ-specific damage. The testicular necrosis produced by Cd is the product of damage to the testicular blood vessels (Gunn et al., 1963) as is the ability of the metal to damage sensory ganglia (Gabbiani et al., 1967b). Placental and ovarian necrosis produced by Cd in rodents may also relate to a fundamental vascular insult (Chiquoine, 1965; Kar et al., 1959). Schroeder et al. (1970) examined the effects of vasoactive substances on the blood pressure of Cd-treated, male Long-Evans rats. Those animals which had received Cd (10 ppm of Cd in drinking water for 2-4 mo) had higher blood pressures than control animals, but exhibited depressed responsiveness to the vasopressor effects of norepinephrine and angiotensin. From these findings, the authors concluded that Cd hypertension may be accompanied by diminished vascular reactivity. Thind et al. (1969) studied the responsiveness of the thoracic aorta from young New Zealand White male rabbits made hypertensive by six to eight weekly injections of Cd. They found the contractile ’ This work wassupportedby Public Health ServiceEnvironmentalToxicology TrainingGrant 2TOl ESOOO71-06. Appreciationis expressed to CibaLaboratoriesfor providing angiotensinamide. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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response to angiotensin to be significantly decreased in aortic strips from the hypertensive animals. In recent studies on Cd-hypertensive dogs and rabbits, Thind (1974) has observed a decreased responsiveness of the isolated carotid artery from Cd-hypertensive dogs to angiotensin, norepinephrine, and serotonin. Similar changes were observed with angiotensin acting on the isolated rabbit aorta; the response of this preparation to norepinephrine, however, was unaltered from that of normotensive controls. Measurement of the wall thickness of the vessels used in these in vitro studies revealed a similar species difference. Rabbit aortic wall thickness was unchanged whereas in the dog carotid artery this value was increased by the Cd treatment. These observations leave unanswered the question of whether a causal relationship exists between Cd-induced hypertension and changes in vascular reactivity. In the present study, the administration to male and female Sprague-Dawley-derived and female hooded Long-Evans rats of the dosage schedule of Cd acetate employed by Schroeder et al. (1966) produced no increase in blood pressure in any of the Cd-treated animals as reported by Porter et al. (1974). It was decided to conduct studies on the vascular reactivity of these Cd-treated rats, to determine if altered vascular responsiveness to vasoactive substances is dependent upon the existence of the hypertensive state following Cd administration, as indicated by the reports of others referenced above. METHODS
Animals. Female Sprague-Dawley-derived rats (Laboratory Supply Co., Indianapolis), 91-94 days of age, were employed in this study. Animals were housed in stainless steel community cages in temperature (22-24°C) and light (14-10 light-dark) controlled rooms and allowed free access to laboratory chow (Allied Mills, Chicago) and distilled water. Indirect blood pressure measurements were made on lightly ether-anesthetized rats by the tail-cuff method of Friedman and Freed (1949). Preliminary experiments showed no significant difference between blood pressures recorded while rats were under light ether anesthesia as compared to animals under pentobarbital anesthesia. Animals were allowed 5 days to acclimate to the laboratory environment and diet, and two control blood pressure recordings at 2-day intervals were taken on all animals prior to Cd administration. Arterial blood pressure responseof the Cd-treated rat to various autonomic agonists andantagonists. A control group of 12 female, 91-day-old Sprague-Dawley-derived rats
received saline (2.0 ml/kg, ip) while a similar group of 12 animals received Cd (2.0 mg Cd/kg, ip) as the acetate. On day 21 following this initial injection, a second dose of Cd (1.0 mg Cd/kg, ip) or saline (1.0 ml/kg, ip) was administered. From days 14-27, following the second administration of Cd, these animals were employed for determination of blood pressure responses to various autonomic drugs. The drugs and dose levels administered included: epinephrine HCl (0.5 pg/kg in 0.01 N HCl); norepinephrine bitartrate (0.5 pg/kg in 0.01 N HCl); acetylcholine iodide (0.1 pg/kg in 0.01 N HCI); isoproterenol HCI (0.01 ,ug/kg in 0.01 N HCI); atropine sulfate (0.5 mg/kg in 0.9 % NaCl solution); and propranolol HCI (1 .Omg/kg in 0.9 % NaCl solution). Animals were anesthetized with DialR-urethane solution (0.6-0.8 ml/kg, ip). Each animal was then secured on a rat board, a tracheal cannula inserted, the left femoral vein
CADMIUM
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14.5
cannulated (PE 20 tubing) for intravenous administration of drugs, and the right carotid artery cannulated (PE 90 tubing) for direct, continuous blood pressure recording employing a P-1000-A pressure transducer and a Physiograph. The carotid cannula contained sodium heparin (100 USP units/ml.) Drugs were injected via the femoral vein in volumes of 0.07-0.09 ml and washed in with 0.10 ml of 0.9 % NaCl solution. Initial systolic blood pressures were between 110 and 150 mmHg. In a few animals where the systolic pressure fell below 90 mmHg during administration of the drug sequence, 5 % bovine albumin was infused until the pressure was restored to greater than 90 mmHg. The sequence of drug administrations was as follows: I. epinephrine; 2. norepinephrine; 3. acetylcholine; 4. isoproterenol (drugs l-4 were then repeated in the same sequence); 5. atropine; 6. acetylcholine; 7. propranolol; 8. isoproterenol; 9. epinephrine; 10. norepinephrine. The control administration of 0.9 % NaCl solution or 0.01 N HCl(0.2 ml) caused insignificant changes in blood pressure. Following the injection of each drug, the maximum rise or fall in blood pressure was recorded. The next injection was not made until the blood pressure had returned to the value obtained before the drug administration and had stabilized. For each animal the blood pressure change following epinephrine, norepinephrine, acetylcholine, and isoproterenol was taken as the average change for the first two injections of each of these drugs, prior to the administration of atropine and propran0101. The blood pressure change following atropine and propranolol was based on a single administration of these drugs to each animal. The mean maximum rise or fall in blood pressure caused by each drug was then compared for animals in the control and Cd-pretreated groups by means of Student’s t test. EfSect of Cd pretreatment of the rat upon the reactivity of aortic smooth muscle to various agonists. A control and a treatment group (16 rats each) of female 94-day-old,
Sprague-Dawley-derived rats received saline (2.0 ml/kg, ip) or Cd (2.0 mg Cd/kg, ip) as the acetate, respectively. On day 21 following this initial injection, a second dose of Cd (1.0 mg Cd/kg, ip) or saline (1.0 ml/kg, ip) was administered. From days 9-37 following the second administration of Cd, studies were conducted on aortic strips from control and Cd-treated animals to determine if the reactivity of this smooth muscle preparation was modified by Cd treatment. Dose-response relationships for the following agents were determined on each aortic strip: tyramine HCl (10-6-10-3 M); angiotensin amide (10-9-10-6 M); epinephrine HCl (10-9-10-5 M); and barium (10V6-IO-” M). All solutions remained stable throughout the period of experimentation, under the conditions of storage employed. Barium was kept at room temperature (22-24”C), while all other drugs were refrigerated (4--5°C). Angiotensin was adjusted to pH 2 with 0.01 N HCl to assure its stability (Thymann, 1968). Tyramine and epinephrine were also adjusted to pH 2. Barium solution was prepared from barium hydroxide which was then adjusted to pH 6 with 2.0 N and 0.01 N HCI. Cd-treated and control animals were ether anesthetized after which the descending thoracic aorta was rapidly excised and placed in cold, oxygenated Krebs-Henseleit solution (Krebs, 1950). The aorta was then cut along a loose spiral with microdissecting scissors by the method of Furchgott and Bhadrakom (1953). This spiral was then cut into two segments of 2 cm each which were mounted in a muscle bath containing 75 ml of Krebs-Henseleit solution. There was no significant difference in the reactivity of the
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upper and lower portions of the aortic strip as measured by the response to epinephrine. The contractile force of aortic strips was measured isometrically with a Grass Force-displacement Transducer FT03C and recorded on a Grass model 5 Polygraph. One gram of tension was placed on all strips and the strips allowed to equilibrate for 2 hr before beginning the drug sequence. After the strips had remained in the bath for 105 min, fresh bathing solution was added and the first dose-response relationship started 15 min later. All drug additions were cumulative with each progressive concentration being added at the peak of the previous response. Following each dose-response measurement, fresh bathing solution was added and the strips allowed to return to baseline tension over a 25-min period. At this time fresh solution was again added and the next dose-response measurement was started 5 min later. The bath temperature was maintained at 37.5”C and the pH at 7.3-7.5. A gas mixture of 95 % O2 and 5 % COz was bubbled through the bath continuously. A volume of 0.1 ml of drug solution was added at each concentration step of every dose sequence except at the 3 x 10m4M and 1 x 10m3 M concentrations of barium. In these instances it was necessary to add 1.0 ml of the barium solutions to the 75-ml muscle bath due to the limited solubility of barium. RESULTS Arterial blood pressure response of the Cd-treated rat to various autonomic agonists and antagonists. Weekly recordings of blood pressures (indirect) following Cd administration disclosed no significant differences between Cd-treated and control rats prior to the use of these animals in subsequent studies of blood pressure responses to various autonomic drugs (Porter et al., 1974). Data in Table 1 show the blood pressure response to various autonomic drugs elicited upon their administration to Cd-treated and TABLE 1 ARTERIAL
BLOOD PRESSURE RESPONSE OF THE CADMIUM-TREATED TO VARIOUS AUTONOMIC AGONISTS AND ANTAGONISTS“
RAT
Systolic blood pressure change (mmH8 +- SE) Drug and dose Epineprhine (0.5 fig/kg) Norepinephrine (0.5 Dug/kg) Acetylcholine (0.1 pg/kg) Isoproterenol(O.01 pg/kg) Atropine (0.5 mg/kg) Propranolol(1 .Omg/kg)
Control T21f 2 (8)b 131f 3 (8) $23+ 2 (8) $18f4(8)
Treated T21+ 1 (7) NS’ f22 + 1 (7)d $16+ 2 (7)d $13+ 3 (7) NS
$21 + 5 (8)
J12 + 2 (7) NS
$34It 5 (7)
$34+ 4 (6) NS
a Sprague-Dawley-derived rats received cadmium (2.0 mg Cd/kg and 1.0 mg Cd/kg, ip) as the acetate, or saline (2.0 ml/kg and 1.0 ml/kg, ip) 21 days apart. The blood pressure responses of control and cadmium-treated animals to intravenously administered autonomic drugs were determined starting 14 days after the second dose of cadmium. * Number of animals per group. c NS=Not significantly different (p > 0.05) relative to saline controls. d Significantly different (p < 0.05) relative to saline controls.
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control rats. A significant (p < 0.05) decrease in the vascular reactivity to norepinephrine and acetylcholine was seen in the Cd-treated animals. A substantial, though not significant, reduction in blood pressure response was also seen in the Cd-treated animals following isoproterenol and atropine. Responses to epinephrine and propran0101 did not differ for the control and Cd-treated groups. Effect of Cd pretreatment of the rat upon the reactivity of aortic smooth muscle to various agonists. Aortic strip dose-response curves for angiotensin, epinephrine.
lJJ .05t( I” w : 2
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1. Effect of cadmium pretreatment of the rat on contractility of thoracic aortic strips to angiotensin. Female, Sprague-Dawley-derived rats received cadmium (2.0 kg Cd/kg and 1.0 mg Cd/kg, ip) as the acetate, or saline (2.0 ml/kg and 1.Oml/kg, ip) 21 days apart. The response to angiotensin of replicate aortic strips from 10 control and 10cadmium-treated animals was measured starting 9 days after the second dose of cadmium. Standard errors are represented by vertical bars. FIG.
IO-*
10-S CONCENTRATION
OF
10-T EPINEPHRINE
(rndos
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FIG. 2. Effect of cadmium pretreatment of the rat on contractility of thoracic aortic strips to epinephrine. See Fig. 1 for experimental conditions. The response to epinephrine of replicate acrtic strips from 10 control and 10 cadmium-treated animals was measured starting 9 days after the second dose of cadmium. Standard errors are represented by vertical bars.
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PORTER,MOYAANDBOUSQUET
CONCENTRATION
OF BARIUM
( molss/litrr)
FIG. 3. Effect of cadmiumpretreatmentof the rat on contractility of thoracic aortic stripsto barium.SeeFig. 1 for experimentalconditions.Theresponse to bariumof replicateaorticstripsfrom 10 control and 10cadmium-treated animalswasmeasured starting9 daysafter the seconddoseof cadmium.Standarderrorsarerepresented by verticalbars. .16cti +I 0
.14-
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ii
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FIG. 4. Effect of cadmiumpretreatmentof the rat on contractility of thoracicaortic stripsto tyramine.SeeFig. 1 for experimentalconditions.Theresponse to tyramineof replicateaortic strips from 10control and 10cadmium-treated animalswasmeasured starting9 daysafter theseconddose of cadmium.Standarderrorsarerepresented by verticalbars.
barium, and tyramine are shown in Figs. 14. A general feature of these responsesis a tendency toward a shifting of each line to the right along the dose axis, and a lower maximum response of the aortic strips from the Cd-treated animals to the several agonists, when compared to strips from control rats. DISCUSSION
The intravenous administration of vasoactive substancesto Cd-treated and control rats demonstrated a significantly (p < 0.05) decreased vascular responsiveness, as
CADMIUM
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determined by the magnitude of the blood pressure response, to norepinephrine and acetylcholine and a substantial (28 and 42x), though not significant, decrease in vascular responsiveness following isoproterenol and atropine in the Cd-treated animals. No difference between control and treated animals was apparent following epinephrine or propranolol. Studies involving the direct addition of vasoactive substances to aortic strips. showed a decreased sensitivity of such strips from Cd-treated animals to angiotensin, epinephrine, barium, and to the indirect-acting agonist, tyramine. The possibility exists that many of the pathophysiological actions of Cd are related to the interaction of Cd with body stores of essential ions, especially calcium and zinc. Specificity of the toxic actions of Cd are difficult to assess in view of the fact that such toxicity may be inhibited by several other metals. For example, the testicular necrosis produced by Cd may be blocked by zinc (Parizek and Zahor, 19X$ selenium (Kar et aE., 1960), and manganese and cobalt (Gunn and Gould, 1970). Increased levels of calcium in drinking water have been reported to suppress Cd-induced hypertension in rats (Schroeder et al., 1967). Friberg et al. (1971) reported one of the major toxic signs of the Cd-induced “Itai-itai” disease to be weakening of the bone structure, which strongly suggests calcium depletion. It is well documented that calcium ion is the agent activating contractile elements in both skeletal and smooth muscle fibers (Hudgins and Weiss, 1968; Hurwitz et al., 1973). Hurwitz et al. (1973) reported that a specific intracellular pool of calcium supports the contractile response to a significant degree in the main pulmonary artery of the rabbit. If the same is true of the rat, depletion of such an intracellular pool of calcium by Cd might lead to a decreased contractile response of the vasculature. Regardless of the precise mechanism(s) involved, the present findings suggest that a decreased vascular reactivity to both vasoconstrictor and vasodilator substances follows Cd administration to the Sprague-Dawley-derived rat. It is a major finding of this study that the ability of Cd treatment of the rat to alter vascular sensitivity to vasoconstrictor and vasodilator agents was shown to be independent of the ability of the metal to induce hypertension. REFERENCES AXELSSON, B., AND PISCATOR, M. (1966). Renal damage after prolonged An experimental study. Arch. Environ. Hlth. 12, 360-373. CHIQUOINE, A. D. (1965). Effect of cadmium chloride on the pregnant
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F. H. (1941). The chronic
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G., BAIC, D., AND DEZIEL, C. (1967b). Studies on tolerance and ionic antagonism for cadmium or mercury. Can. J. Physiol. Pharmacol. 45,443-450. Gum, S. A., GOULD, T. C., AND ANDERSON, W. A. D. (1963). The selective injurious response of testicular and epididymal blood vessels to cadmium and its prevention by zinc. Amer. J. Pathol. 42,685-702. GUNN, S. A., GOULD, T. C., AND ANDERSON, W. A. D. (1964). Effect of zinc on cancerogenesis by cadmium. Proc. Sac. Exp. Biol. Med. 115,653-657. GUNN,S. A., AND GOULD, T. C. (1970).Cadmiumand other mineral elements.In The Testis A. D. Johnson,W. R. Gomer, and N. L. Van Demask,pp. 377-481.Academic Press,Inc., New York. HUDGINS, P. M., AND WEISS, G. B. (1968).Differential effectsof calciumremovalupon vascular smooth muscle contraction induced by norepinephrine, histamine and potassium.J. Pharmacol.Exp. Ther. 159,91-97. HURWITZ, L., FITZPATRICK, D. F., DEBBAS, G., AND LANDON, E. J. (1973). Localization of calciumpump activity in smoothmuscle.Science179, 384-386. KAR, A. B., DAS, R. P., AND KARKUN, J. N. (1959).Ovarian changesin prepubertalrats after treatment with cadmiumchloride. Acta Biol. Med. Ger. 3, 372-379. KAR, A. B., DAS, R. P., AND MUHERJJ, F. N. I. (1960).Prevention of cadmiuminducedchanges in the gonadsof rat by zinc and selenium-a study in antagonismbetweenmetalsin the biological system. Proc. Natl. Inst. Sci. India B26 (Suppl. 40), 40-50. KREBS, H. (1950).Body sizeand tissuerespiration. Biochem.Biophys.Acta 4,249-269. PARJZEK, J., AND ZAHOR, Z. (1956).Effect of cadmiumsaltson testiculartissue.Nature(London) GABBIANI,
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M. C., MIYA, T. S.,AND BOUSQUET, W. F. (1974).Cadmium:Inability to inducehypertensionin the rat. Toxicol. Appl. Pharmacol.27,692-695. SCHROEDER, H. A., KROLL, S. S., LITTLE, J. W., LIVINGSTON, P. O., AND MYERS, M. A. G. (1966).Hypertensionin rats from injection of cadmium.Arch. Environ. H&h. 13,788-789. SCHROEDER, H. A., NASON, A. P., AND BALASSA, J. J. (1967). Trace metals in rat tissues as influencedby calciumin water. J. Nutr. 93, 331-335. SCHROEDER, H. A., BAKER, J. T., HANSEN, N. M., JR., SIZE, J. G., AND WISE, R. A. (1970). Vascularreactivity of rats alteredby cadmiumand a zinc chelate.Arch. Environ. Health 21, PORTER,
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G. S., STEPHAN, K. R., AND BLAKEMORE, W. S. (1969). Vascular responsiveness in cadmium-inducedhypertension.Fed. Proc. 28,403ABS. THJND, G. S., DARREMAN, G., STEPHAN, K. R., AND BLAKEMORE, W. S. (1970). Vascular reactivity and mechanicalpropertiesof normal and cadmium-hypertensiverabbits. J. Lab. Clin. Med. 76, 560-568. THJND, G. S. (1974).Blood vesselwall characteristicsin experimentalhypertension.Angiology THJND,
25,752-763. M. (1968).The stability of angiotensinamide in aqueous solution. DanskTidsskrift Farmaci 42,225-234. WILSON, R. H., DEEDS, F., AND Cos, A. J., JR. (1941).Effects of continuedcadmiumfeeding. J. Pharmacol.Exp. Ther. 71,222-235. THYMANN,
