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Abstract for IMM92044

Immunotoxicity of Morphine Sulfate in Female B6C3F1 Mice

CASRN: 64-31-3
Chemical Formula: C17H19NO3.1/2H2O4S
Molecular Weight: 668.76
Report Date: December 1992

Abstract

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.

 

Morphine sulfate represents the prototype opiate antagonist which binds to the opiate receptor and produces biological effects on several systems of the body. Its actions as an analgesic and tolerance to this effect is well documented.1 Other systems of the body affected include the autonomic nervous system,2,3 cardiovascular and pulmonary systems,4,5,6 hepatobiliary system7,8,9,10 and the immune system. 11,12,13,14,15,16

The studies reported here represent an holistic investigation of morphine's effect on the immune system. Since heroin works through the same mechanism as morphine and also has been implicated in immune suppressive activity, these studies will help in understanding the relationship between drugs of abuse such as heroin and immune suppression.

Morphine sulfate was nominated for investigation by the National Institute of Drug Abuse based on several diverse studies that indicated that it is immunosuppressive.

Morphine sulfate was administered by slow release pellet. In a range-finding study, a time course for immunosuppression was performed using the IgM spleen antibody-forming cell response as the indicator of immunosuppression. The peak time for immunosuppression occurred when the antigen was administered 24 hours after implant. In these same studies, complete tolerence to the morphine action occurred by day 5 after implantation. Morphine levels reaching 1610 ng/ml were obtained within 24 hours of pellet implant. A request was made to the National Institute of Drug Abuse for 3 pellet dose levels of morphine. As a part of the range-finding investigation, a dose-response study was performed which measured morphine levels and corticosterone levels as well as the IgM spleen antibody-forming cell response. The pellet sizes used were 8, 25 and 75 mg pellets. The studies showed that 24 hours after implant morphine levels were 904 ng/ml, while mice implanted with 25 and 75 mg dose p! ellets had mean blood levels of 1299 and 1610 ng/ml. The bioavailability of morphine in these pellets was not linear and the release on corticosterone was very similar for all three pellet sizes.

Executive Summary Table 1 (ES-1) shows a summary of the standard toxicology studies. The primary objective of the toxicology studies was to characterize the toxicological profile produced by morphine. The implantation of morphine did not result in any mortality. Implantation of morphine pellets which produced blood levels as high as 1600 ng/ml caused changes in body weight and body weight gain, changes in hematological parameters and in serum chemistries.

Morphine implantation also increased the blood levels of corticosterone which is believed to have an effect on some of the parameters measured. Body weights of mice implanted with morphine have up to a 10% decrease in body weight. Changes in hematology, as manifested in an increase in erythroid elements, may be related to hemoconcentration, while the decrease in leukocyte numbers may be related to the cortiscosterone-induced apoptosis of lymphocyte and the activation of the sympathetic nervous system which may increase neutrophil output of the bone marrow. Elevation of SGPT, serum albumin and total protein are indicative of liver damage.

Executive Summary Table 2 (ES-2) summarizes the immunology studies of mice treated with morphine sulfate. Morphine sulfate, administered by a slow release subcutaneously implanted pellet to female B6C3F1 mice, is associated with several changes in immune function. The changes in immune function are also associated with elevation of corticosterone levels and may be the major mediator of the immune changes. Spleen and thymus weights and spleen cellularity are decreased by as much as 42%. Spleen cell differentials showed a 50.4% decrease in the percent of Ig+ cells and an increase in the percent of Thy 1.2+ (26.6%), CD4+ (17.3%) and CD8+ (27.3%) cells. Because of the decrease in spleen cellularity, there was an overall decrease of the B cells and T cells in the spleen. The IgM AFC response to the T-dependent antigen sRBC was decreased 57% when expressed as specific activity, i.e. AFC/106 spleen cells, and 72% when expressed on a per spleen basi! s. The spleen proliferative response to T cell and B cell specific mitogens or to allogeneic cells (MLR) were not markedly altered as was the abililty of the T cells to produce cytotoxic T cells (CTL). Innate immunity was markedly altered as seen in suppression of macrophage activity, NK cell activity and decreased serum C3 levels. The decreased levels of C3 are not believed to be sufficient to be responsible for the decrease in macrophage function. Kupffer cell phagocytosis was suppressed by 51.4%, NK activity by 68.4% and C3 levels by 47.4%. The suppression of NK activity is reversed by naltrexone and RU486 indicating that this action is mediated through the opiate receptor elevating the levels of corticosterone which is the final effector hormone.17 The suppression of Kupffer cell function is reversed by naltrexone and only partially reversed by RU486, indicating that the action is mediated through the opiate receptor and but not completely t! hrough the elevation of corticosterone.18

Executive Summary Table 3 (ES-3) summarizes the host resistance studies in mice treated with morphine sulfate. Implantation of morphine sulfate pellets was associated with decreased resistance in the tumor model, B16F10 melanoma, but increased host resistance to the bacterial pathogenic models, Listeria monocytogenes and Streptococcus pneumoniae. The increase in resistance is not understood and in-depth studies will be needed. In a preliminary study, serum from morphine sulfate-implanted mice did not inhibit the growth of the bacteria in vitro.

The conclusion from these investigations is that morphine sulfate, administered by pellet implant, is associated with elevations in corticosterone that may in part be responsible for effects on selected aspects of the immune system. The most sensitive immune parameters include the antibody response to sRBC, Kupffer cell phagocytosis and NK cell activity. Statistically significant effects were seen at the lowest dose levels of morphine sulfate administered.


References

1 Jaffee, J.H. and Martin, W.R. Opioid analgesics and antagonists in the pharmacological basis of therapeutics, ed. Gilman, Rall, Nies, and Taylor, 485-521, 8th Edition, 1990.

2 Appel, N.M., Kirtsy-Roy, J.A. and Van Loon, G.R. Mu receptors at discrete hypothalmic and brainstem sites mediate opioid peptide-induced increases in central sympathetic outflow. Brain Res., 378:8-20, 1986.

3 May, C.N., Ham, I.W., Heslop, K.E., Stone, F.A. and Mathias, C.J. Intravenous morphine causes hypertension, hyperglycemia and increases sympatho-adrenal outflow in conscious rabbits, Clin. Sci., 75:71-77, 1988.

4 Eckenhoff, J.E. and Oech, S.R. The effect of narcotic and antagonists upon the respiratory and circulation of man. A review. Clin. Pharmacol. Ther., 1:383-524, 1960.

5 Evans, A.G.J., Nassmyth, P.A. and Stewart, H.C. The fall of blood pressure caused by intravenous morphine in the rat and cat. Br. J. Pharmacol., 7:542-552, 1952.

6 Holaday, J.W. Cardiovascular effects of morphine, pethidine, diamorphine and nalorphine on the cat and rabbit. Br. J. Pharmacol., 42:159-178, 1971.

7 Chang, Y.H. and Ho, I.K. Effects of acute and continous morphine administration on serum glutamate oxalacetate transaminase and glutamate pyruvate transaminase activites in the mouse. Biochem. Pharmacol., 28:1373-1377, 1979.

8 Hurwitz, A., Looney, G., Sullins, M. and ben-Zvi, Z. Hepatobilary effects of morphine area mediated in the brain. Hepatology, 12:1406-1412, 1990.

9 Needham, W.P., Shuster, L., Kanel, G.C. and Thompson, M.L Liver damage from narcotics in mice. Toxicol. Appl. Pharmacol., 58:157-170, 1981.

10 Roberts, S.M., Skoulis, N.P. and James, R.C. A centrally-mediated effect of morphine to diminish hepatocellular glutathione. 36:3001-3005, 1987.

11 Bayer, B.M., Daussin, S., Hernandez, M. and Irvin, L. Morphine inhibition of lymphocyte activity is mediated by an opioid dependent mechanism. Neuropharmacol. 29:369-374, 1990.

12 Casellas, A.M., Guardiola, H. and Renaud, F.L. Inhibition by opioids of phagocytosis in peritoneal macrophaages. Neuropeptides, 18:35-40, 1991.

13 Perz-Castrillion, J. Perez-Arellano, J., Garcia-Palomo, J., Jimenez-Lopez, A. and DeCastro, S. Opioids depresses in vitro human monocyte chemotaxis. Immunopharmacology 23:57-61, 1992.

14 Shibinga, N.E.S. and Goldstein, A. Opioid peptides and ipioid receptors in cells of the immune system. Ann. Rev. Immunol., 6:219-249, 1988.

15 Tubaro, E., Borelli, G., Croce, C. Cavallo, G., and Santiangeli, C. Effect of morphine on resistance to infection. J. Infect. Dis., 148:656-666, 1983.

16 Pruett, S.B., Han, Y.C. and Fuchs, B.A. Morphine Suppresses Primary Humoral Immune Response by a Predominantly Indirect Mechanism. J. Pharmacol. and Exp. Ther., 262:923-928, 1992.

17 Frier, D. and Fuchs, B.A. A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. J Pharmacol Exp Ther. 1994 Sep;270(3):1127-33.

18 LeVier, D. G., Brown, R.D., McCay, J.A. Fuchs, B.A., Harris, L.S. and Munson, A.E. Hepatic and splenic phagocytosis in female B6C3F1 mice implanted with morphine sulfate pellets. J Pharmacol Exp Ther. 1993 Oct;267(1):357-63.

Studies


Table ES-1
Summary Table for Toxicology Studies MS-1-1-SC
Parameter Result Maximum Effect Dose Pellet
Level
Comment
Body Weight
Day 1 No Effect
Day 2 Decreased 9.5% 75 mg Dose Dependent
Weight Changes
Days 2-1 Decreased 583% 75 mg Dose Dependent
Organ Weights
Liver Decreased 20% 75 mg Dose Dependent
Spleen Decreased 42.1% 75 mg Dose Dependent
Lungs No Effect
Thymus Decreased 30.6% 75 mg Dose Dependent
Kidney Decreased 16.9% 25 mg Dose Dependent
Hematology
RBCs Decreased 8.1% 25 mg Dose Dependent
Hemoglobin Decreased 8.8% 25 mg Dose Dependent
Hematocrit Decreased 6.3% 8 mg
MCV No Effect
MCH No Effect
MCHC No Effect
Reticulocytes No Effect
Leukocytes Decreased 37.5% 75 mg
Leukocyte Diff Dose Dependent
Lymphocytes Decreased 57% 75 mg Dose Dependent
Neutrophils Increased 486% 75 mg Dose Dependent
Eosinophils No Effect
Serum Chemistries
SGPT Increased 354% 75 mg Dose Dependent
Albumin Increased 25.1% 8 mg
BUN No Effect
Glucose Decreased 27.2% 75 mg Dose Dependent
Total Protein Increased 21.4% 25 mg Dose Dependent
Albumin/Globulin No Effect
Bilirubin Decreased 65.3% 75 mg Dose Dependent
Table ES-2
Summary Table for Immunology Studies MS-1-1-SC
Parameter Results Maximum
Effect
Dose
Pellet Level
Comment
Surface Markers
lg+ Decreased 53.1% 75 mg
Thy 1.2+ Decreased 13.5% 25 mg Based on
CD4+CD8- Decreased 33.3% 75 mg Absolute
CD4-CDB+ Decreased 23.0% 75 mg Values
CD4+CD8+ Decreased 12.2% 75 mg
Spleen IgM Antibody-Forming Cell Response to Sheep Erythrocytes
PFC/106 Cells Decreased -57% 75 mg Dose Dependent
PFC/Spleen Decreased -72% 75 mg Dose Dependent
Proliferation Assays, i.e. Mitogens, Mixed Leukocyte Response
Con A Decreased 28.1% 25 mg Dose Dependent
LPS Increased 17.3% 25 mg Dose Dependent
F(ab')2 + BSF-1 No Effect
Medium Decreased 31.2% 75 mg Dose Dependent
MLR No Effect
Cytotoxic T Lymphocyte Activity
CTL No Effect
NK Cell Activity
100:1 Decreased 55.3% 75 mg Dose Dependent
50:1 Decreased 58.3% 75 mg Dose Dependent
25:1 Decreased 68.4% 75 mg Dose Dependent
RES Function Decreased 51.4% 75 mg Dose Dependent
Liver phagocytosis
Serum C3 Levels Decreased 47.4% 75 mg Dose Dependent
Table ES-3
Summary Table for Host Resistance Studies MS-1-1-SC
Parameter Results Maximum
Effect
Dose
Pellet Level
Comment
B16F10 Melanoma Decreased
Resistance
46% increase in
tumor burden
25 mg Dose dependent
No significant
effects at any
single dose level
Listeria monocytogenes Increased
Resistance
83% reduction in
mortality
8 mg
Streptococcus pneumoniae Increased
Resistance
59% reduction in
mortality
25 & 75 mg