Detoxification or Chemical Defense by Glucuronidation An Undergraduate Biochemistry Experiment Michel Boiret and Alain Marly U.F.R.: S.E.E.-(Chimie) Universitb de Perpignan, 66025 Perpignan Cedex, France
The response of living organisms to environmental chemicals is intensively studied by a number of laboratories (1-5). I t is a highly complex subject, because in the same cell, a delicate balance exists between toxification and detoxification pathways. The aim of this paper is to propose a simple experiment showing students how living cells can defend themselves against xenohiotic aggression. A xenobiotic is an exogenous compound, usually lipid soluble, of no functional value to the organism. Therefore, its continued presence is undesirable, thus toxic (2).The organism will thus endeavor to reduce or suppress this toxicity: it is the detoxication process. Among the possible ways of detoxication, one of the most important is the conjugation of the xenohiotic molecule with a highly polar molecule, or ion, to form a water-soluble conjugate. The xenohiotic conjugated in this way will he rapidly excreted in the extracellular fluid. Glucuronidation is the most wides~readform of coniuea. .. l i o n in mammalian nieraholism. Glucun)nic acid, supplied by uridine d~l)hnsphnten-1)-glucuronic acid (L'DP-(;A), is conjugated with alipid-soluhie xenobiotic to form a watersoluble glucuronide. This reaction (eq 1) is catalyzed by the UDP-glucuronyltransferase EC 2.4.1.17 (UDP-GT).
0
COOH
Benzo(a)pyrene (BPI is a ubiquitous environmental pollutant and its metabolism has been extensively studied (3). BP would therefore have heen a xenohiotic particularly suited to our experiment, but it must be dismissed because i t is a potent carcinogen. We preferred t o use one of its major metabolites, 3-hydroxyhenzo(a)pyrene (3-OH-BP) because previous studies (6)have proved that it is noncarcinogenic. Although the liver seems to he of paramount importance for glucuronidation of xenobiotics, several extrahepatic sites may contribute to the total conjugative capacity. The cellular material chosen is the mouse peritoneal macrophages because i t is an easy system for students to use. The aim of the experiment can be summarized as the demonstration of a system of chemical defense deployed by macrophages when they are challenged by 3-OH-BP dissolved in their culture medium. Prlnclple of the Method The glucuronidation reaction can he written as R-OH + UDP-GA
+ UDP
(1)
where R-OH and R-0-GA represent, respectively, 3-OH-BP and BP-3-glucuronide (Fig. 1). Among the compounds involved in this reaction, only 3-OH-BP and BP-3-glucuronide are fluorophors (7) (Fig. 2).
... .-. .-.::... .. ..
--* .-
400 Figure 1. Structure of 3-OKBP (I); UDPGA(I1): BP-Sglucuronide (Ill). and UDP IIVI.
R-0-GA
BP-3
GLUCURONIDE
....
3-OH-BPCULTURE MEDIUM ...-
500
X nm
Figure 2. Fluorescence spectra of 3-OH-BP ( I w M and BP-3-glucuronide ( I p v solutions in culture medium at pH = 7 4. (A excmtion = 380 nm.)
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.. ... .. :
Incubation timesfmi"
1
than the highly adherent macrophages). The xenobiotic solution and the macrophages can be put in contact between 1 h and 5 d after extraction.
Incubation t i m e s (min) 1.t,
0
Preparationof the Xenoblotic Solution After evaporating 0.2 mL of an alcoholic stock solution of 3-OHBP (0.1 mM), the deposit was dissolved in 20 mL of eultue medium by stirring for 30 min. The concentration of 3-OH-BP in culture medium is about 1@M.
0
ExperimentA At time zero, 1mg of UDP-GA and 5 mg of UDP-GT, both solid, were added to 5 mL of the xenobiotic solution. The reaction mixture was stirred for 5 min and incubated at 37 OC.
t
00
-
-
500
ExperimentB At time zero, the culture medium of a flask containing about 1.5 X lo6 adherent macrophages was replaced by 5 mL of the xenohiotic solution. This reaction flask was incubated at 37 "C.
A nm
-
Fiawe 3. F~U(KBSCBIICB s&ra recorded during lha alucuronidation standard of 3-OKBP (experimem A) and me gluc~ronldationof 3-OKBP by mactophagss (experiment 8).
Two experiments A and B are performed in parallel a t 37 "C from a xenobiotic solution c o m ~ o s e dof culture medium Experiment A is a standard containing 3-OH-BP (1 glucuronidation reaction a s in e q 1because the cosubstrate (UDP-GA) and the enzyme (UDP-GT) are introduced into t h e xenobiotic solution. Experiment B is t h e test of macrophage chemical defense toward 3-OH-BP, as the macrophages are cultured in t h e xenobiotic solution. T h e fluorescence is measured during the two experiments. Then, by comparison, it is possible t o determine t h e part of elucuronidation occurrine in A and B (Fie. 3). T h e glucuronides e x h i k t the characteristic property of beine selectivelv hvdrolvzed in the presence of B-alucuronid a s e " ~3.2.1.31 ~ T ~ U Sthe regeneration of 3-OH-BP by 0-glucuronidase treatment is used as a complementary characterization of BP-3-glucuronide.
m.
-
(2).
Chemicals and Apparatus UDP-GA (No.: U 6751), UDP-GT (No.: U 3626). andb-glucuronidase (No.: 105-2003)were obtained from Sigma. 3-OH-BP was obtained from NCI Standard Repository, Bethesda. The culture medium was the Gibco 199 medium with Hanck's salts and L-glutamine supplemented with 10%fetal calf serum (pH 7.4). All spectra were recorded on a Perkin Elmer spectrofluorometer with an excitation wavelength of 380 nm. Cell c u m e Peritoneal macrophages of untreated mice of indifferent filiation, age, and sex were easily obtained by following the standard extraction procedure of Conrad (8).The extraction medium was the culture medium at 4 OC and the number of extracted macrophages per mouse routinely amounted to 1-2 million cells. Such a cell suspension was introduced in a sterile culture flask (Costar 25 mL). This flask wasmaintained 1h a t 37 'C (in order to removeany cells other 1010
Journal of Chemical Education
8-Glucuronidase Treatment When the experiment A and B were finished, each reaction culture medium (about 5 mL) was introduced into a vial containing 1.000 units of 8-elucuronidase ourified. The hvdrolvsis reached cbmpletion withinulo min at 37 "C Sampling and SpectrofluorometricAnalysis The analytical procedure was identical for experiments A and B, and the 0-glucuronidasetreatment. At different incubation times, a sample of about 2 mL of culture medium was taken from the reaction flask, then introduced in a quartz cuvette end the fluorescence spectrum was recorded. Then, the sample was replaced into the reaction flask. This analytical procedure was routinely performed within 2 min. Results and Discussion Figure 3 illustrates t h e changes in fluorescence during experiments A and B. It shows t h a t in both cases, 3-OH-BP
,\
ROPHAGE
MEDIUM
3 -OH-BP
BP-3- Glucuronide
xmobiotir
Conj~gate
Lipid -Soluble
Water Soluble
Figure 4. Scheme of chemical defense by peritoneal macrophages of mice toward 3-OKBP.
is progressively transformed into BP-3-glucuronide. Indeed, both fluorescence spectra recorded a t the end of the reaction (#4 in both A and B) are characteristic of BP-3-glucuronide (Fig. 2). Furthermore, the glucuronide nature of the conjueate formed is confirmed bv. . B-elucuronidase treatment. The comparison between experiments A and B thus enable us to conclude that the chemical defense of ~eritoneal mouse macrophages in the presence of 3-OH-BP is exclusively based on elucuronidation. Thissystem, ;epresented in Figure 4, can be formulated in three s t e ~ s R-OH-BP : u ~ t a k e(step 1). intracellular elucuronidation (step 2), and BP-3-gluc&onide excretion (&ep 3). The residual intracellular BP-3-glucuronide (Fig. 4) is responsible for the difference of intensity between spectra 4A and 4B (Fig. 3). The chemical defense by glucuronidation seems particularly efficient because it was possible to carry out up to five successive experiments on the same population of macrophages without affecting their viability.
-
Extension
If the instructor deems i t necessary, the results can be quantitated. Thue, the rate of glucuronidation can be determined in versus parameters such as initial 3-OH-BP concentration, number of macrophages, and influence of an inhibitor (7). Moreover, one may emphasize the fact that normal and cancerous tissues present major differences in glucuronidation rates (9). Literature Cited (1) Pelkunen.0,; Nebert. D. W. Phnrmoco! RPU.198'2,34,189. 121 Dutlon, G. J. "Glucumnidation of Drugs and Other Compounds:" CRC Baca Raton, FI. 19RO I31 Gelhuin, H. V. Physiol. Rau. 1980,60.1107. I 4 Nemoto, N. In '"PolycyclicHydroearhonr and Canoer;" Gelbin. H. V.: Ts'o, P. 0. P.. Eds.: Acadernic:New York. 1981: Vol 3. Chap 5. (5) Caldwell. J. Life Sci. 1973.24.571.
I61 Wir!ocki.P.G.;Chang,R.L.;Woad,A.W.;Leuin,W.;Yagi,H.;Hemandez,O.;Msh.H. D.; Dsnsette. P.M.; Jerina, D. J.; Conney, A. H. CencarRss. 1977.37.2608. 17) Boiret, M.: Marty, A : DeumiO, M. Anal Biochsm. l986,153,132. 181 Conrad,R. E. In"ManualofMacmphageMcthadologi~He-witeH.B.;Holden,H. T.;Bollanti, J. A ; Ghaffar, A,, Eda.; Dekker:New Yark, 1981: Chap 1. 191 Cohcn.G. M.;Gibhy, E. M.;Mehts,R. Nature 1981,291,662,
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