Toxicology Lerters, 51 (1990) 227-231

227

Elsevier

TOXLET

023 12

Inhibitory effects of carcinogenic tryptophan pyrolysis products on phytohemagglutinin-induced blast transformation of human lymphocytes

Hiroyuki

Yanagisawa*

and Osamu Wada

Department of Hygiene und Preventive Medicine, Faculty of Medicine, University ofTokyo, Tokyo (Japan) (Received

5 October

(Revision

received

(Accepted

1989) 11 October

13 November

1989)

1989)

Key words: Carcinogenic

heterocyclic

amines: Phytohemagglutinin;

Blast transformation

of human

lymphocytes

SUMMARY In order to evaluate human

the effect of carcinogenic

cells, we examined

methylimidazo

[4,5:jjquinoline

dipyrido[l.2-u:3’,2’-d]imidazole 3-amino-1-methyl-SH-pyrido[4,3-h] formation

induced

I .O-10 PM, respectively. inhibitory

(Glu-P-2).

The concentrations

However,

effect on lymphocyte

on human (PHA).

stimulated of Trp-P-l

the other carcinogenic mitogenesis

on the stimulussreaction

heterocychc

lymphocyte

added at the final concentration

2-amino-3-

Both Trp-P-l

and Trp-P-2

and Trp-P-2

causing amines

exhibited of

50% inhibition examined

amines

culture medium

an inhibi-

I .O-5.0 and were 3.1 and

did not show any

of 10 PM. Viabilities heterocyclic

of 10 PM to the lymphocyte

and

(4 x lo5 cells/ml) blast trans-

heterocyclic

even at a final concentration

2-amino-

(Trp-P-l)

by PHA at the final concentration

cytes were more than 90% (n = 3 x 5) when 5 kinds of carcinogenic respectively

system of

amines including

3.amino-1,4-dimethyl-SH-pyrido[4,3-blindole

indole (Trp-P-2) proliferation

amines

1,2-u:3’,2’-d)imidazole (Glu-P-l),

(IQ), 2-amino-6_methyldipyrido[

by 0.125% phytohemagglutinin

tory effect on the lymphocyte 5.1 PM, respectively.

heterocyclic

the effect of 5 kinds of carcinogenic

of lympho-

(0.1 ml) were

(4 x IO5 cells/O.9

ml).

INTRODUCTION

The diet is recently regarded as one of the most important factors related to the incidence of human cancers [l]. Various kinds of mutagenic heterocyclic amines have * Current address and correspondence: H. Yanagisawa, University

School of Medicine,

0378-4274/90/$3.50

Renal

Division,

Box 8126, 660 South Euclid Avenue,

@ 1990 Elsevier Science Publishers

Internal

Medicine,

Washington

St. Louis, MO 63110, U.S.A.

B.V. (Biomedical

Division)

22x

been reported to be present in cooked foods, cigarette smoke condensate and fried egg patties [224]. The majority of those mutagens have shown carcinogenic effects in animal experiments [2], and are now supposed By using the dialysate and plasma of patients correlation

between

human

cancers

to be dietary with uremia,

and carcinogenic

carcinogens. we have examined

heterocyclic

amines.

the

We re-

ported that several carcinogenic heterocyclic amines were present in the dialysate and plasma of patients with uremia, and that humans were actually exposed to several carcinogenic heterocyclic amines including 3-amino-l,4-dimethyl-5H-pyrido[4,3-h]indole (Trp-P-l) and 3-amino-I-methyl-5H-pyrido[4,3-hlindole (Trp-P-2) [559]. More recently, we reported that carcinogenic tryptophan pyrolysis products inhibited cycle-oxygenase activity in human platelet aggregation induced by ADP, collagen and sodium arachidonate [lo]. In this investigation, in order to investigate further effects of carcinogenic heterocyclic amines on the stimulus-reaction system of human cells, we examined the influence of 5 kinds of carcinogenic heterocyclic amines on human lymphocyte blast transformation induced by phytohemagglutinin (PHA). As a result, we demonstrated that among the 5 carcinogenic heterocyclic amines only tryptophan pyrolysis products showed an inhibitory effect on lymphocyte mitogenesis. MATERIALS

AND METHODS

Trp-P- 1 acetate, Trp-P-2 acetate, 2-amino-6_methyldipyrido[ 1,2-a:3’,2’-dimidazole (Glu-P-l), 2-aminodipyrido[l,2-a:3’,2’-d]imidazole (Glu-P-2) and 2-amino-3methylimidazo[4,5Tflquinoline (IQ) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Heparin was obtained from Novo Industri A/S (Copenhagen, Denmark). The lymphocyte blast transformation research kit (Japan Immunoresearch Laboratories Fluorescence Blast Transformation Test: JIMRO FBT) was kindly provided by Otsuka Assay Laboratories (Tokushima, Japan). Preparation and culture of human lymphocytes and the lymphocyte blast transformation test by PHA were performed by using JIMRO FBT. Five healthy male volunteers were studied. The ages of the healthy males ranged from 26 to 32 years (mean 29.4 + 2.4 (SD) years, n = 5). Peripheral blood lymphocytes were prepared from heparinized blood of 5 subjects by Ficoll-Conray (Otsuka Assay Laboratories) gradient centrifugation. The lymphocytes separated were washed with 50 mM phosphate-buffered saline (PBS) 3 times and suspended at the concentration of 1 x IO6 cells/ml in RPM1 1640 containing 10% fetal calf serum (FCS) (culture medium). Finally, 5 kinds of carcinogenic heterocyclic amines (IQ, Glu-P- I, Glu-P-2. Trp-P-l and Trp-P-2) were adjusted to various concentrations (10, 25, 50, 100 PM) in the culture medium. The lymphocyte suspension (4 x IO5 cells/O.4 ml) was divided into each well of 24-well cultured plates, and then preincubated for 15 min with 100 ,uI of various concentrations of carcinogenic heterocyclic amine solutions or 100 ,~l of the culture medi-

229

urn in the case of the control. by adding

The preincubated

500 ,~l of 0.125% PHA in the culture

samples

were cultured

medium

for 72 h at 37°C with 5%

in triplicate

CO2 in a humidified atmosphere. The lymphocyte blast transformation test by PHA was carried out by using ethidium bromide fluorescence assays based on JIMRO FBT [11,12]. The 72-h cultured lymphocytes were transferred into new plastic tubes from each well of the culture plates and then centrifuged at 2500 rpm at room temperature for 10 min. After the removal of supernatant from the tubes, 2 ml of 0.125% sodium dodecylsulfate (SDS) was added to each tube, and the tubes were then allowed to stand for 30 min at room temperature. Next, each tube was allowed to stand for 15 min in the presence of 2 ml ethidium bromide (7.5 pg/ml). Finally, lymphocyte reactivity to PHA was determined by fluorescence intensities (excitation at 525 nm, emission at 600 nm) using a Hitachi fluorescence spectrophotometer 650-60 (Hitachi, Tokyo, Japan). The percent inhibition of lymphocyte mitogenesis by carcinogenic heterocyclic amines was calculated with the formula below: X-Y % Inhibition =x-z x 100, where X = fluorescence intensities of lymphocytes stimulated by PHA, Y = fluorescence intensities of lymphocytes by PHA in the presence of carcinogenic heterocyclic amines, and Z = fluorescence intensities of lymphocytes without stimulation by PHA. Percent inhibition was expressed as a mean value of triplicate measurements. The mean and SD were calculated on the basis of 5 experiments. RESULTS

AND DISCUSSION

The optimal concentration of PHA was determined in the range of 0.01-l .O% (0.5 ml) by using lymphocytes (4 x lo5 cells/O.5 ml) from 3 healthy male volunteers. The strongest fluorescence intensities of lymphocytes stimulated by PHA were obtained at the concentration of 0.084.15%. The PHA concentration of 0.125% was therefore used for the stimulation of lymphocytes, as described previously [12]. Viabilities of lymphocytes were checked by adding trypan blue to an aliquot of human lymphocytes (4 x IO5 cells/O.9 ml) cultured for 72 h with 5 kinds of 100 ,uM carcinogenic heterocyclic amines (0.1 ml) using a phase-difference microscope. Viabilities of lymphocytes examined were more than 90% (n = 3 x 5) in all cases. To investigate the effect of carcinogenic heterocyclic amines on the stimulus-reaction system of human cells, the effects of IQ, Glu-P-1, Glu-P-2, Trp-P-l and Trp-P-2 were examined on human lymphocyte blast transformation induced by PHA. As shown in Table I, both Trp-P-l and Trp-P-2 strongly inhibited lymphocyte mitogenesis to PHA at the final concentration of 1.O-5.0 and 1.&lo ,uM, respectively. However, the other carcinogenic heterocyclic amines (IQ, Glu-P-1 and Glu-P-2) examined did not exhibit any inhibitory effect on lymphocyte blast transformation to PHA even at the final concentration of 10 ,uM. Trp-P-l showed the inhibitory effect more

230 TABLE’1 INHIBITORY

EFFECTS

LYMPHOCYTE

OF CARCINOGENIC

PROLIFERATION

INDUCED

HETEROCYCLIC

AMINES

ON THE

HUMAN

BY PHYTOHEMAGGLUTININ

Final

“/; Inhibition

concentration Trp-P- I

Trp-P-2

IQ

95.6k4.4

0.42

5.0

9X.7+9.2

49.2 & 3.2

2.5

31.4+ 7.8

32.254.0

9.8k2.5

27.71X.3

(/lM) IO.0

I .o Trp-P-1 indole; 3’2.d]

= 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; IQ

=

Trp-P-2

2-amino-3-methylimidazo[4,5;1]quinolme;

imidazole;

Glu-P-2 = 2-aminodipyrido[

Glu-P-1

1,2-a:3’.2’-d]imidazole.

I.0

Glu-P- I

Cl&P-?

0.8 f 3.2

0.6 & 3.6

= 2-ammo-l-methyl-SH-pyrido[4,3-h]=

2-amino-6-methyldipyrido-[1,2-u:

Values represent

mean k SD for 5 ex-

periments.

strongly than Trp-P-2 and almost completely inhibited the lymphocyte response to PHA at the final concentration of 5 ,uM. The concentrations of Trp-P-l and Trp-P-2 causing 50% inhibition (I(&) of lymphocyte proliferation induced by PHA were estimated to be approximately 3.1 and 5.1 ,vM, respectively. Although it is unknown that the inhibitory effect of Trp-P-l and Trp-P-2 is actually taking place in vivo and is related to the carcinogenic action of these carcinogens, the fact that the carcinogens to which humans are actually exposed exhibit in vitro an inhibitory effect on the stimulussreaction system of human cells would be very interesting. Further studies are necessary for elucidating the mechanisms of the inhibitory effect on human lymphocyte blast transformation induced by PHA. ACKNOWLEDGEMENTS

We wish to thank Dr. Hisanori ma, Japan), for kindly providing FBT).

Matsumoto, Otsuka Assay Laboratories (Tokushithe lymphocyte blast transformation kit (JIMRO

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