The differential response of isolated intestinal crypt and tip cells to the inductive actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Chem.-Biol. Interactions, 22 (1978) 199--209

199

© Elsevier/North-Holland Scientific Publishers Ltd.

THE D I F F E R E N T I A L RESPONSE OF ISOLATED INTESTINAL CRYPT AND TIP CELLS TO THE INDUCTIVE ACTIONS OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN

CAROL

M. S C H I L L E R and G E O R G E

W. L U C I E R

Laboratory of Environmental Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research TrianglePark, N.C. 27709 (U.S.A.)

(Received November 7th, 1977) (Revision received March 8th, 1978) (Accepted March 20th, 1978)

SUMMARY

The cell specific induction of uridine diphosphate(UDP)-glucuronyltransferase by 2,3,7,8-tetrachlorodibenzo-p
INTRODUC~ON

2,3,7,8-Tetrachlorodibenzo-p
200 dation, and its acute, chronic and cumulative toxicity have generated considerable concern a b o u t this potential environmental health hazard. A striking feature of the well~tocumented toxicity of TCDD [1,4-6] is the minute quantity of TCDD required to produce pathologic effects. The biochemical lesions underlying the observed toxicity of TCDD are n o t known, although activities of several enzymes are altered after non-lethal doses of TCDD to rodents [7,9]. Recently, it has been demonstrated that UDP-glucuronyltransferase and aryl hydrocarbon hydroxylase are induced in microsomes isolated from rat intestine, liver, kidney and lung [10]. These two enzyme systems are closely related in their inductive response to TCDD and are the most sensitive biochemical indicators known of TCDD tissue concentrations [ 11,12]. Induction of aryl hydrocarbon hydroxylase by TCDD has been studied in a variety of cell types from both primary cultures and established cell lines. While these studies did n o t provide a positive correlation between c y t o t o x i c i t y and induction, they did provide evidence that certain sensitive cell lines m a y be useful in a bioassay for detecting small amounts of TCDD; inducibility of UDP-glucuronyltransferase and aryl hydrocarbon hydroxylase in undifferentiated hepatoma cell lines is detectable at a TCDD concentration of 10 -t4 M [ 1 3 , 1 4 ] .

The intestinal epithelium is composed of several distinct cell types with characteristic morphologic and enzymic complements which reflect specific metabolic functions. Cell proliferation is confined to the cells of the crypt regions, while the absorptive cells are located at the tips of the villi. As the epithelial cells differentiate during migration from the crypts to the villous tips, from which the cells are eventually shed, the ability to divide is lost. The absorptive cells contain well-developed microvilli that are associated with high levels of hydrolase activities. While the intestine has been used for numerous studies to measure differential responses to hormones, the susceptibility of these cells to environmentally significant bioactive c o m p o u n d s such as TCDD has n o t been evaluated. In the present study, the TCDD-mediated induction of UDP-glucuronyltransferase is monitored in cells isolated from the tip and crypt regions of the intestine. The sensitivity of the isolated tip and c r y p t cells to this TCDDmediated induction of UDP-glucuronyltransferase is determined by measuring concentrations of [ 14C] TCDD in each of these cell types over a time course. M A T E R I A L S AND M E T H O D S

Animals

Adult female specific pathogen-free rats (Charles River, CD strain) were used in these experiments. On the day of treatment the rats weighed approx. 200 g (10-week-old). The rats were fasted for 16 hr. before TCDD was administered as a single oral dose (8 ug/kg) in approx. 0.5 ml of corn oil. The control rats received 0.5 ml of corn oil. The animals were fed ad libitum after the oral dosing until the time of sacrifice by decapitation. Fasting had

201 no significant effect on the UDP-glucuronyltransferase activities. When ['4C]TCDD was used, it was administered as described above at a specific activity of 148 mCi/mmol (approx. 0.73 uCi/animal).

Preparation o f cells Intestinal cells were isolated from segments of the small intestine by a low-amplitude, high-frequency vibration method [15,16]. This procedure was modified by including 0.4 mM dithiothreitol in the isotonic saline, pH 7.4 for washing the intestinal lumen free of its contents. Also, the segments were shaken for an initial 30 s and then for 3 five-minute intervals, in 0.14 M NaC1 containing 5 mM EDTA, pH 7.4, in order to isolate the tip, intermediate and c r y p t cell fractions in sequence. The everted segments were dilated by insufflation to expose the c r y p t regions before the final interval. The cells were sedimented b y centrifugation at 500 g for 5 min. and then resuspended in 0.9% NaC1. This procedure was repeated to wash the cells and then the cells were resuspended in assay buffer. Preparation of the cells was monitored routinely by trypan blue d y e exclusion as well as by measuring the characteristic marker enzymes for each cell type. Trypan dye exclusion indicated that the cells were approx. 70% viable. Aliquots of the cells from each preparation were fixed in a glutaraldehyde-formaldehyde solution and were processed for electron microscopic analysis [17]. The cells were examined under a Philips EM 300 at the 10--14 000 × range. In addition, aliquots of the cells from the [14C]TCDD treated animals were stored in a freezer until they could be analyzed by oxidation in a Harvey Biological Oxidizer, R.J. Harvey Instrument Corp., Hillsdale, N.J. All methods of sample preparation, oxidation and liquid scintillation counting were identical to those described previously [ 18]. Assay me thods All enzyme assays were performed under optimal cofactor and substrate concentrations. ~-Glucuronidase (EC 3.2.1.31) was determined by a modification of the method of Lucier and McDaniel [19] with p-nitrophenyl-fl-Dglucuronide as the substrate in a final volume of 0.4 ml. UDP-glucuronyltransferase (EC 2.4.1.17) was determined spectrophotometricaUy [20] by using 0.9 mM p-nitrophenol, 0.8 mM UDP-glucuronic acid, 10 mM MgC12, and Triton X-100 treated cells in a total volume of 0.4 ml. After 10 min of incubation, the reaction was stopped by the addition of 1.0 ml glycine buffer, pH 10.4 [21], and the p-nitrophenol released measured at 405 nm. Alkaline phosphatase (EC 3.1.3.1) was determined spectrophotometrically at 405 nm with a Beckman Acta III recording spectrophotometer b y using 15 mM p-nitrophenylphosphate and 0.1 M diethanolamine adjusted to pH 9.8 with 1.0 N HC1 [22]. Acid phosphatase (EC 3.1.3.2) was determined by an end-point spectrophotometric m e t h o d [22]. The assay mixture contained 5.5 mM p-nitrophenylphosphate and 50 mM sodium citrate, pH 4.8. At the end of the incubation, the pH was adjusted with 0.1 N NaOH before the samples were read at 405 nm. D N A synthesis was determined in

202 intact cells by the incorporation of [aH]thymidine into DNA isolated and counted by a previously described m e t h o d [23]. 2.0-ml aliquots of the cell suspension in Krebs-Ringer-bicarbonate buffer were incubated in the presence of 12.5 #mol glucose, 22 m~ bovine serum albumin and 44 nmol [3H] thymidine, 125 ~Ci/gmol. The reaction was terminated after 20 rain by the addition of trichloroacetic acid. Protein was measured with the phenol reagent [24] and bovine serum albumin as a standard. Data are expressed per mg of cell protein as the intestinal cells tend to aggregate which makes accurate counts of cell n u m b e r difficult. Chemicals TCDD (Lot No. 851-144 II; purity > 99%) was supplied as a gift by the Dow Chemical Co., Mich. ~4C-labeled TCDD was synthesized by the condensation of 4,5
Characterization o f the isolated cells The differential vibration m e t h o d was used for the sequential removal of the intestinal epithelium from the lamina propria of the villi. Evidence for the gradual removal of the cells (cells from the tip of the villi are dislodged first and those of the crypt last) is based on the decline of the specific activities of a brush border enzyme, alkaline phosphatase, and the increase of rate of [aH]thymidine incorporation in these cells in the subsequent fractions. In contrast, the lysosomal enzyme activity, acid phosphatase, which is associated with both cell types remained relatively constant (Table I). As shown in Table I, there is a 4--5-fold difference both in the specific activity of the tip cell marker enzyme alkaline phosphatase and the crypt cell marker activity [aH]thymidine incorporation found in the isolated tip and crypt cells. These observations are in agreement with those previously reported [16,23]. Further evidence that the epithelium was selectively TABLE I DISTRIBUTION OF MARKER ENZYMES IN INTESTINAL CELLS Cell

Alkaline phosphatase (mU/mg)

Acid phosphatase (mU/mg)

DNA synthesis (mU/mg)

Tip

172 125 36.0

29.4 35.1 23.9

1.15 2.08 5.54

Intermediate Crypt

203 removed from underlying connective tissue was provided by examining the resulting cells and remaining tissue, before and after insufflation, under a light microscope (not shown). The characteristic cellular architectures of these cells appeared grossly intact. The morphological examination in connection with the biochemical studies mentioned above (Table I), v~ich were applied in each experiment, assured that a reasonably good separation of these types of cells had been obtained.

Distribution o f intestinal UDP-glucuronyltransferase Glucuronidation of p-nitrophenol was measured using the tip cells isolated from duodenal, jejunal and ileal portions of the small intestine. As shown in Fig. 1 there is a 10-fold difference in the UDP-glucuronyltransferase activity between the proximal and distal proteins of the female rat small intestine. Therefore, only the duodenal segments of the female rats were used in the subsequent time~ourse experiments. Time-course effects o f TCDD on UDP-glucuronyltransferase in intestinal crypt and tip cells Adult female rats were fasted for 16 hr and then fed ad libitum after receiving 0.73 ~Ci of [~4C]TCDD (148 mCi/mmol) as a single oral dose at 8 ~g/kg. The LDs0 value for a single oral dose of TCDD is approx. 100 ~g/kg [1]. p-Nitrophenol glucuronidation was measured 0, 3 and 10 h and 1, 3 and 5 days after treatment. At each time period, tip and crypt cells were harvested from the duodenal segments of 9 rats pooled in groups of three as three separate samples. Basal activities of crypt and tip cells were approx. 6.5 and 13 nmol p-nitrophenol conjugated per min/mg protein, respectively (Fig. 2). UDPglucuronyltransferase activities were approximately the same as control values three hrs following TCDD treatment. However, in the 10 hr group tip 30

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IC

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Fig. 1. UDP-Glucuronyltransferaseactivity in proximal and distal portions of the rat small intestine.

204

B

24

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r ~ Crypt Tip

I

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Fig. 2. T i m e - c o u r s e e f f e c t s o f T C D D o n U D P - g l u c u r o n y l t r a n s f e r a s e activity in intestinal c r y p t and tip cells N=3. Each value r e p r e s e n t s the m e a n + 1 S.D. Details o f the experim e n t a l design are given in the materials and m e t h o d s s e c t i o n and discussed in the text.

._o

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~" I0

Days after TCDD treatment

Fig. 3. R a t i o s o f tip to c r y p t cell specific activities for U D P - g l u c u r o n y l t r a n s f e r a s e aftel" TCDD t r e a t m e n t . Calculations are based o n the e x p e r i m e n t described in Fig. 2 and l-epresent a matched-pair comparison.

205 cell activity increased by 20% compared to a 50% elevation of activity in c r y p t cells. The differential induction between the intestinal cell types became more pronounced by 1 day after TCDD administration; 200% increase in crypt cell activity and 50% increase in tip cell activity. Ratios of tip to crypt cell activities were significantly l o w e r than control from 1--5 days after TCDD treatment although both cell types still contained elevated UDP-glucuronyltransferase levels (Fig. 3). Furthermore, the ratio at one day after treatment was significantly lower than in the five day group. Tip to crypt UDP-glucuronyltransferase specific activity ratios during the time-course experiment demonstrate that the control ratio was 2.1, the ratio decreased to 0.9 by 1 day after treatment followed by an increase to approximately 1.3 at the 3- and 5-day time periods.

Time-course o f [~4C] TCDD incorporation and retention in intestinal tip and retention in intestinal tip and crypt cells There was a negative correlation between the time-course of UDP-glucuronyltransferase induction and the ~4C-activity content measured in these cells (Fig. 4). For example, tip cell ~4C concentrations were more than crypt cell concentrations at all time points although crypt cells were more sensitive -

oCrypt eTip

80-

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4C

20

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Time ( ~ y s )

Fig. 4. Time-course of [ ' * C ] T C D D incorporation and r e t e n t i o n in intestinal c r y p t and tip cells. Each animal was given 0.73 uCi of [3*C]TCDD (148 m C i / m m o l ) as described for Fig. 2.

206 to TCDD-mediated induction of UDP-glucuronyltransferase. Tip cell concentrations decreased from 50 dpm/mg cellular protein to 20 dpm/mg from 3 to 24 h following TCDD administration. However, in the 3- and 5~lay sample, tip cell 14C-activity levels increased to approx. 75 dpm/mg protein. This increase probably reflects reexposure of intestinal cells to TCDD via the biliary route. No attempts were made at this time to determine the subcellular distribution of ~4C-activity in isolated crypt and tip cells or the chemical nature of the 14C-labeled material. DISCUSSION The method used in this study for the isolation of the tip and crypt cells combines both chemical (EDTA~belation) and mechanical, (low-amplitude, high-frequency vibration) methods [15]. In particular, this method has the advantage that in a short period of time both cell types can be harvested separately in reasonably good, reproducible yields [25]. A combination of morphologic [15, 23, 27] and enzymic [16, 23] criteria established that the cells in our preparations were well separated. These isolated intestinal cells have been used advantageously for the study of the TCDD-mediated induction of UDP-glucuronyltransferase in both the tip and crypt cells. Recent studies have shown that several microsomal drug metabolizing enzymes can be induced in rat intestinal tissue. Male rats treated with a single oral dose of TCDD (25 #g/kg) were observed to have markedly increased levels (at least 3-fold) of intestinal microsomal biphenyl 2-hydroxylase, benzpyrene hydroxylase and UDP-glucuronyltransferase activities three days after treatment [ 10]. Intestinal microsomes isolated from rats pretreated with 3-methylcholanthrene and phenobarbital also contained biphenyl 2hydroxylase activity approximately 3 times higher than the control activity [26]. Since the intestinal epithelium contains both differentiated (tip) and undifferentiated (crypt) cell populations, the present study was designed to evaluate the inducibility characteristics of each cell type with UDPglucuronyltransferase as the enzymic indicator of induction. The greater sensitivity of the crypt cells to the TCDD-mediated effects on the intestine suggests a more general sensitivity of undifferentiated cells to inductive agents. Evidence for this greater sensitivity of the crypt cells to the actions of TCDD is provided by the fact that UDP-glucuronyltransfemse induction in crypt cells occurs prior to the elevation of tip cell enzyme activity (Figs. 2 and 3) and in the negative correlation between the level of UDP-glucuronyltransferase induction and the measured levels of [ ~4C]TCDD in these same cell preparations (Fig. 4). Estimates of the time required for the transit of the crypt cells to the tips of the villi and to be sloughed off are usually between 48 and 72 h [23, 28]. During this period, the crypt cells lose their capacity to proliferate and become adapted to perform absorptive functions. The levels of UDPglucuronyltransferase increase 2-fold in this transition period (Fig. 2). The observed time necessary for the appearance of marked UDP-glucuronyl-

207 transfemse induction in the tips cells correlates well with the crypt to tip migration time of 2--3 days (Figs. 2 and 3). A recent study reported the 3-methylcholanthrene-mediated induction of the intestinal mixed-function oxidases in tip cells. However, in this study induction was measured in the tip cells four days after treatment [29] which is longer than the crypt to tip migration time. In addition, these researchers used cells labeled in vivo with [SH]thymidine to verify a transition period of 48 h in their preparation. Because of the choice of time after treatment for these measurements, any interpretation as to the contribution o f cell type to the inductive process is limited. It should be pointed out that, unlike the mixed-function oxidases, UDP-glucuronyltransferase is not a cytochrome P-450~iependent enzyme system and does not require heme biosynthesis for activity. An interesting observation derived from the results of our study is that the levels of 14C-activity in both cell types were the highest at 3--5 days after treatment (Fig. 4). This observation is consistent with the results of an earlier pharmacokinetic study [30] in which the clearance of a single oral dose of 1 ~g of [14C]TCDD/kg is monitored. This study demonstrated that most of the body burden was found in the liver and fat, and that the ~4C-activity found in the liver was unchanged TCDD. However, preliminary work indicates that materials other than TCDD constitute a significant fraction of 14C-activity in the feces. That [14C]TCDD is metabolized is also consistent with the fact that ~4C-activity is excreted in the urine of rats [30] and in the urine and expired air from rats [1]. Therefore, while much of the administered ~4C-activity is localized in the liver and the ~4C-activity in the liver has been identified as [14C]TCDD, small amounts of polar metabolites may be formed in the liver and readily excreted in the bile and urine, or excreted in the bile and subsequently reabsorbed. The elevated levels of 14C-activity observed in the isolated cells in our study may, in part, be a reflection of this enterohepatic circulation of TCDD or a more polar metabolite. REFERENCES 1 Environmental Health Perspectives 1973 Experimental Issue 5, Proceedings of NIEHS Conference on Toxicity of Chlorinated Dibenzo-p-dioxins and Dibenzofurans, April 2--3. 2 Report on 2,4,5-T 1971 A Report on the Panel on Herbicides of the President's Science Advisory Committee, Government Printing Office, Washington, D.C. 3 Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency 1971 Government Printing Office, Washington, D.C. 4 H. Bauer, K.G. Schulz and U. Spiegelberg, Berufliche vergiftungen bei der herstillung yon chlorophenol verbidungen, Arch. Gewerbepathol. Gewerbehyg., 18 (1961) 538. 5 G.R. Higginbotham, A. Huang, D. Firestone, J. Verret, J. Ress and A.D. Campbell, Chemical and toxicological evaluations of isolated and synthetic chloro derivatives of dibenzo-p-dioxins, Nature, 220 (1968) 702. 6 N.P. Buu-Hoi, P.H. Chang, G. Sesque, M.C. Azum-Gelade and G. Saint-rut. Organs as targets of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) intoxication, Naturwissenschaften, 59 (1972) 174.

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209 28

J.J. Deren, Development o f intestinal structure and function, in: C.F. Code (Ed.), Handbook of Physiology, Vol. 3, Sect. 6, Williams & Wilkins, Baltimore, 1968, pp. 1099--1123. 29 H. Hoensch, C.H. Woo, S.B. Raffin and R. Schmid, Oxidative metabolism of foreign compounds in rat small intestine: Cellular localization and dependence on dietary iron, Gastroenterology, 70 (1976) 1063. 30 J.Q. Rose, J.C. Ramsey, T.H. Wentzler, R.A. Hummel and P.J. Gehring, The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat, Appl. Pharmacol., 36 (1976) 209.