Toxicology Letters, 63 (1992) 313-319
313
TOXLET 02816
In vitro effects of ‘designer’ amphetamines on human peripheral blood mononuclear leukocytes proliferation and on natural killer cell activity
Lyne Gagnona*’, France Lacroixb, Joyce Ghan” and Harpal S. Buttara “Bureaux of Drug Research and bChemicalSafety, Sir Frederick Banting Research Centre, Health Protection Branch, Ottawa, Ontario (Canada)
(Received 29 June 1992) (Accepted 26 August 1992) Key words: Amphetamines;
‘Designer’ amphetamines; Lymphocyte proliferation; Natural killer cell; Im-
munomodulation SUMMARY Human peripheral blood mononuclear leukocytes (PBML) proliferation was measured in the presence or absence of amphetamines. Proliferation in response to T-cell mitogen PHA was suppressed from 22 to 34% by d- and dl-amphetamine, respectively, contrarily to l-form which did not affect proliferation of PHAstimulated PBML. The ‘designer’ amphetamines appeared to be more potent inhibitors of PBML proliferation induced by both PHA and PWM stimulation than those of the racemic and isomeric forms of amphetamine. A wide variation was seen in the suppressive actions of the ‘designer’ amphetamines, and the mean percentages of suppression varied from 12 to 45% compared with the control values. 4-Propoxyamphetamine (4-PA) was found to be the most active among the ‘designer’ drugs. In vitro effects of d-, 1- and dl-amphetamine were also studied on natural killer (NK) cell activity. A marked increase in the NK cell activity was observed only in the presence of very low concentrations (lo-l2 to 10-i’ M) of dl-amphetamine, however, the activity of the NK cell remained within the control limits in the presence of d- or l-forms. The findings suggest that the abuse of amphetamines, especially the ‘designer’ drugs, may adversely affect the activity of immunoregulatory cells and might lead to a compromised immune system in amphetamine abusers.
Correspondence to: Dr. Harpal S. Buttar, Life Sciences Division, Bureau of Drug Research, Sir F.G. Banting Research Centre, Tunney’s Pasture, Ottawa, Ontario, KlA 0L2, Canada. ‘Present address: Dr. Lyne Gagnon, National Research Council of Canada, Biology Division, Cell Signals
Group, Building M-54, Montreal Road, Ottawa, Ontario, KlA 0R6, Canada.
314
INTRODUCTION
Dextroamphetamine, methamphetamine and dl-amphetamine are used for treating psychoneurotic illness involving depression and narcolepsy, attention-deficit disorders and exogenous obesity. Abuse and addiction have increased correspondingly with the number of synthesized amphetamine derivatives. In fact, d&hetamine and methamphetamine are the two most widely abused clandestinely synthesized drugs [I]. 4Substituted or ‘designer’ amphetamines also have significant potential as drugs of abuse and addiction [224]. Substances of abuse, such as cocaine [.5,6], opiate [7], marijuana [8,9], and ethanol [6] were found to affect immunoregulatory cells. The purpose of the present investigation was to determine the effects of d-, I- and dlamphetamines and ‘designer’ amphetamines on human peripheral blood mononuclear leukocytes (PBML) proliferation and on natural killer (NK) cell activity. MATERIALS AND METHODS
Amphetamines
Racemic dl-amphetamine HCl was purchased from Sigma (St. Louis, MO), isomeric d- and 1-~phetamine sulphate were obtained from Smith-Kline & French (Montreal, Quebec). The 4-substituted or ‘designer’ amphetamines were synthesized as hydrochIoride salts at the Bureau of Drug Research, Health Protection Branch, Ottawa. The 4-substituted amphetamines used in this study included 4-benzyloxyamphetamine (4-BA), 4-ethoxyamphetamine (4-EA), 4-hydroxyamphetamine (4-HA), 4methoxyamphetamine (4-MA), and 4-propoxyamphetamine (4-PA). All compounds were initially dissolved in distilled water and then further diluted in appropriate concentrations in tissue culture medium. Controls contained the vehicle alone. Cell preparations
Human peripheral blood mononuclear leukocytes (PBML) were isolated from heparinized blood by density gradient centrifugation on Ficoll-Hypaque (Pharmacia, Montreal, Quebec) gradients [IO]. By Wright-Giemsa staining they represented 75 90% lymphocytes and l&25% monocytes. After three washes in phosphate-buffered saline (PBS), they were resuspended in appropriate concentrations in RPM1 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories, NY). Proliferation assay
Mitogen-induced lymphocyte proliferation was assayed by measuring the uptake of [3H]thymidine by PBML during the last 6 h of a 72-h culture in the presence of phytohemagglutinin A (PHA, 3.6 pglwell, Difco Laboratories, Detroit) and the last 6 h of a 7-days culture in the presence of 0.3 j&well of pokeweed mitogen (PWM) (Gibco, Burlington, Ontario) with or without various concentrations of drugs. The cells were incubated at 37°C 95% OX+ 5% CO,. Cells were harvested at 72 h or 7 days
315
and total associated radioactivity was measured in counts per minute (CPM). Results were expressed as percent suppression as follows: percent suppression = 1 -
CPM after drug incubation x 100 CPM of controls >
NK cell assay
Cytotoxic activity of PBML was measured using a 4 h “Cr-release microassay [ 111. Target cells (K562, erythroleukemia cell line) were labelled with “Cr (Na,“CrO,, New England Nuclear), washed three times with PBS and distributed into microtiter wells at a concentration of 5 x lo3 cells per well. PBML were then added to half of the wells at a concentration of 2.5 x lo5 cells per well, giving an effector to target cell ratio of 50: 1. Alternate wells contained medium alone. Appropriate concentrations of amphetamines were prepared in the medium MEM and added to additional sets of microtiter wells containing PBML-targets or medium-targets culture. Cytotoxicity was assayed by measuring the “Cr content of culture supernatants. Cytotoxic activity was expressed as percent specific “Cr release as follows: percent specific “Cr release = (FF I ii) where RE represents experimental release, RS, spontaneous release of 51Cr.
x 100 release, and RT, total
Statistical analysis
The data are presented as means ? standard error from six different experiments. The means were compared using unpaired Student’s t-test. The differences were considered significant when P < 0.05.
40
1
l
* PXO.01
OJ, 6 AMPHETAMINE
,
I
7
6
CONC.
I-LOG,,
[Ml1
Fig. 1. Effects of d-, 1-, and dl-amphetamine after 72 h exposure on the proliferation of human PBML in response to the T.-cell mitogen phytohemagglutinin A (PHA).
316
60 z ti +I 50 f UJ 40 I z
0 4-BA m 4-EA [XI 4-HA ISI 4-MA :P:s **P&O1
0.01 4-SUBSTITUTED
0.1 AMPMTAMINES
1.0 I~GIML)
Fig. 2. Effects of 4-substituted amphetamines after 72 h exposure on the proliferation of human PBML in response to the T-cell mitogen phytohemagglutinin A (PHA).
RESULTS
Proliferation in response to T-cell mitogen PHA was evaluated in the presence or absence of isomeric or racemic forms of amphetamines. Figure 1 shows that l-amphetamine does not affect proliferation of PHA-stimulated PBML. At concentrations of 10m6M both d- and dl-forms suppressed significantly the PHA-induced proliferation ranging from 22% to 34%, respectively. The suppression was not due to the toxic effect of amphetamines on PBML as indicated by Trypan blue exclusion. The concentration-response study revealed that increasing concentrations of ‘designer’ amphetamines suppressed human lymphocyte proliferation in response to the T-cell mitogen PHA. The results indicated a peak activity at a concentration of 0.1 j.&nl (Fig. 2). The ‘designer’ amphetamines appeared to be more potent inhibitors than dl-, d- or l-amphetamine, and the mean percentages of suppression varied from 12 to 41% depending upon the doses and drugs used. Their rank order of potency was: 4-PA>4-MA>4-HA>4-EA>4-BA. The ‘designer’ amphetamines also suppressed the human PBML proliferation caused by the B-cell pokeweed mitogen (PWM). Once again, the maximal suppression was observed at concentrations of 0.1 &ml (Fig. 3) after 7 days exposure. The average magnitude of suppression varied from about 20 to 45%. The rank order of suppressive activity of ‘designer’ amphetamines on the PWM-induced proliferation of PBML was: ~-PA>~-MA>~-BA>~-HAz~-EA. Figure 4 shows that dl-amphetamine was effective in enhancing the NK cell activity directed against target cells K562, as indicated by a significantly greater percentage of “Cr release. This augmentation of the NK cell activity occurred at very low concentrations, such as lo-l2 to lo-” M, whereas at higher concentrations dl-amphetamine
317
Cl 4-BA a
jz 2 B s: t-p c *p
313
TOXLET 02816
In vitro effects of ‘designer’ amphetamines on human peripheral blood mononuclear leukocytes proliferation and on natural killer cell activity
Lyne Gagnona*’, France Lacroixb, Joyce Ghan” and Harpal S. Buttara “Bureaux of Drug Research and bChemicalSafety, Sir Frederick Banting Research Centre, Health Protection Branch, Ottawa, Ontario (Canada)
(Received 29 June 1992) (Accepted 26 August 1992) Key words: Amphetamines;
‘Designer’ amphetamines; Lymphocyte proliferation; Natural killer cell; Im-
munomodulation SUMMARY Human peripheral blood mononuclear leukocytes (PBML) proliferation was measured in the presence or absence of amphetamines. Proliferation in response to T-cell mitogen PHA was suppressed from 22 to 34% by d- and dl-amphetamine, respectively, contrarily to l-form which did not affect proliferation of PHAstimulated PBML. The ‘designer’ amphetamines appeared to be more potent inhibitors of PBML proliferation induced by both PHA and PWM stimulation than those of the racemic and isomeric forms of amphetamine. A wide variation was seen in the suppressive actions of the ‘designer’ amphetamines, and the mean percentages of suppression varied from 12 to 45% compared with the control values. 4-Propoxyamphetamine (4-PA) was found to be the most active among the ‘designer’ drugs. In vitro effects of d-, 1- and dl-amphetamine were also studied on natural killer (NK) cell activity. A marked increase in the NK cell activity was observed only in the presence of very low concentrations (lo-l2 to 10-i’ M) of dl-amphetamine, however, the activity of the NK cell remained within the control limits in the presence of d- or l-forms. The findings suggest that the abuse of amphetamines, especially the ‘designer’ drugs, may adversely affect the activity of immunoregulatory cells and might lead to a compromised immune system in amphetamine abusers.
Correspondence to: Dr. Harpal S. Buttar, Life Sciences Division, Bureau of Drug Research, Sir F.G. Banting Research Centre, Tunney’s Pasture, Ottawa, Ontario, KlA 0L2, Canada. ‘Present address: Dr. Lyne Gagnon, National Research Council of Canada, Biology Division, Cell Signals
Group, Building M-54, Montreal Road, Ottawa, Ontario, KlA 0R6, Canada.
314
INTRODUCTION
Dextroamphetamine, methamphetamine and dl-amphetamine are used for treating psychoneurotic illness involving depression and narcolepsy, attention-deficit disorders and exogenous obesity. Abuse and addiction have increased correspondingly with the number of synthesized amphetamine derivatives. In fact, d&hetamine and methamphetamine are the two most widely abused clandestinely synthesized drugs [I]. 4Substituted or ‘designer’ amphetamines also have significant potential as drugs of abuse and addiction [224]. Substances of abuse, such as cocaine [.5,6], opiate [7], marijuana [8,9], and ethanol [6] were found to affect immunoregulatory cells. The purpose of the present investigation was to determine the effects of d-, I- and dlamphetamines and ‘designer’ amphetamines on human peripheral blood mononuclear leukocytes (PBML) proliferation and on natural killer (NK) cell activity. MATERIALS AND METHODS
Amphetamines
Racemic dl-amphetamine HCl was purchased from Sigma (St. Louis, MO), isomeric d- and 1-~phetamine sulphate were obtained from Smith-Kline & French (Montreal, Quebec). The 4-substituted or ‘designer’ amphetamines were synthesized as hydrochIoride salts at the Bureau of Drug Research, Health Protection Branch, Ottawa. The 4-substituted amphetamines used in this study included 4-benzyloxyamphetamine (4-BA), 4-ethoxyamphetamine (4-EA), 4-hydroxyamphetamine (4-HA), 4methoxyamphetamine (4-MA), and 4-propoxyamphetamine (4-PA). All compounds were initially dissolved in distilled water and then further diluted in appropriate concentrations in tissue culture medium. Controls contained the vehicle alone. Cell preparations
Human peripheral blood mononuclear leukocytes (PBML) were isolated from heparinized blood by density gradient centrifugation on Ficoll-Hypaque (Pharmacia, Montreal, Quebec) gradients [IO]. By Wright-Giemsa staining they represented 75 90% lymphocytes and l&25% monocytes. After three washes in phosphate-buffered saline (PBS), they were resuspended in appropriate concentrations in RPM1 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories, NY). Proliferation assay
Mitogen-induced lymphocyte proliferation was assayed by measuring the uptake of [3H]thymidine by PBML during the last 6 h of a 72-h culture in the presence of phytohemagglutinin A (PHA, 3.6 pglwell, Difco Laboratories, Detroit) and the last 6 h of a 7-days culture in the presence of 0.3 j&well of pokeweed mitogen (PWM) (Gibco, Burlington, Ontario) with or without various concentrations of drugs. The cells were incubated at 37°C 95% OX+ 5% CO,. Cells were harvested at 72 h or 7 days
315
and total associated radioactivity was measured in counts per minute (CPM). Results were expressed as percent suppression as follows: percent suppression = 1 -
CPM after drug incubation x 100 CPM of controls >
NK cell assay
Cytotoxic activity of PBML was measured using a 4 h “Cr-release microassay [ 111. Target cells (K562, erythroleukemia cell line) were labelled with “Cr (Na,“CrO,, New England Nuclear), washed three times with PBS and distributed into microtiter wells at a concentration of 5 x lo3 cells per well. PBML were then added to half of the wells at a concentration of 2.5 x lo5 cells per well, giving an effector to target cell ratio of 50: 1. Alternate wells contained medium alone. Appropriate concentrations of amphetamines were prepared in the medium MEM and added to additional sets of microtiter wells containing PBML-targets or medium-targets culture. Cytotoxicity was assayed by measuring the “Cr content of culture supernatants. Cytotoxic activity was expressed as percent specific “Cr release as follows: percent specific “Cr release = (FF I ii) where RE represents experimental release, RS, spontaneous release of 51Cr.
x 100 release, and RT, total
Statistical analysis
The data are presented as means ? standard error from six different experiments. The means were compared using unpaired Student’s t-test. The differences were considered significant when P < 0.05.
40
1
l
* PXO.01
OJ, 6 AMPHETAMINE
,
I
7
6
CONC.
I-LOG,,
[Ml1
Fig. 1. Effects of d-, 1-, and dl-amphetamine after 72 h exposure on the proliferation of human PBML in response to the T.-cell mitogen phytohemagglutinin A (PHA).
316
60 z ti +I 50 f UJ 40 I z
0 4-BA m 4-EA [XI 4-HA ISI 4-MA :P:s **P&O1
0.01 4-SUBSTITUTED
0.1 AMPMTAMINES
1.0 I~GIML)
Fig. 2. Effects of 4-substituted amphetamines after 72 h exposure on the proliferation of human PBML in response to the T-cell mitogen phytohemagglutinin A (PHA).
RESULTS
Proliferation in response to T-cell mitogen PHA was evaluated in the presence or absence of isomeric or racemic forms of amphetamines. Figure 1 shows that l-amphetamine does not affect proliferation of PHA-stimulated PBML. At concentrations of 10m6M both d- and dl-forms suppressed significantly the PHA-induced proliferation ranging from 22% to 34%, respectively. The suppression was not due to the toxic effect of amphetamines on PBML as indicated by Trypan blue exclusion. The concentration-response study revealed that increasing concentrations of ‘designer’ amphetamines suppressed human lymphocyte proliferation in response to the T-cell mitogen PHA. The results indicated a peak activity at a concentration of 0.1 j.&nl (Fig. 2). The ‘designer’ amphetamines appeared to be more potent inhibitors than dl-, d- or l-amphetamine, and the mean percentages of suppression varied from 12 to 41% depending upon the doses and drugs used. Their rank order of potency was: 4-PA>4-MA>4-HA>4-EA>4-BA. The ‘designer’ amphetamines also suppressed the human PBML proliferation caused by the B-cell pokeweed mitogen (PWM). Once again, the maximal suppression was observed at concentrations of 0.1 &ml (Fig. 3) after 7 days exposure. The average magnitude of suppression varied from about 20 to 45%. The rank order of suppressive activity of ‘designer’ amphetamines on the PWM-induced proliferation of PBML was: ~-PA>~-MA>~-BA>~-HAz~-EA. Figure 4 shows that dl-amphetamine was effective in enhancing the NK cell activity directed against target cells K562, as indicated by a significantly greater percentage of “Cr release. This augmentation of the NK cell activity occurred at very low concentrations, such as lo-l2 to lo-” M, whereas at higher concentrations dl-amphetamine
317
Cl 4-BA a
jz 2 B s: t-p c *p
