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Polychlorinated terphenyls and intestinal transport in mice
{h.n Pharma~ . 1977 I~,! 8. pp 43 to 46 Pergamon Press Printed in Great Britain
POLYCHLORINATED TERPHENYLS A N D INTESTINAL TRANSPORT IN MICE DAVID S. MADGE Unit of Applied Zoology, Wye College (University of London), Ashford. Kent TN25 5AH, England (Received 10 October 1976)
Abstract I. Investigations were made on in vitro intestinal absorption of different substrates and fluid transfer in mice following single oral doses of isomers of polychlorinated terphenyls {PCTs). 2. Depending on the dose, D-glucose absorption, but not fluid transfer, was depressed following PC'T-treatment, as was the D-glucose content of tissue homogenates. Malabsorption was generally unrelated to the chlorine content of the PCTs. However, absorption of D-galactose, L-arginine and I,-histidinc. including fluid transfer, remained unaltered. 3. Malabsorption of D-glucose probably resulted from intracellular metabolic effects of PCTs and not malfunction of the D-glucose carrier at the mucosal membrane. 4. Thc histology of the small intestine was unchanged, as was the wet intestinal weight. 5. Absorption experiments using PCTs were compared with previous work on PCBs, and suggested that both malabsorptive effects were similar.
intestine quickly removed, its lumen flushed out with 0.9",, saline solution, and the intestine turned inside out using a slender steel rod. One end of the intestine was ligatured, the intestine filled with 1.25 cm 3 of oxygenated KrebsRinger bicarbonate solution containing the dissolved substrate (serosal fluid), and the other end then ligatured Each sac was put into a 150cm a Erlenmeyer flask containing 25 cm 3 of the same test solution and substrate (mucosal fluid), and the air inside the flask replaced with a mixture of 95% 0 2 + 5°0 CO2. The whole procedure took about 8 min. The flask was then shaken (80 oscillations/min and 2 cm amplitude) in a water-bath at 37C for 1 hr. Following incubation, samples of the serosal and mucosal fluid were analyzed for the appropriate test substrate. The empty sac was washed in saline and the tissue then homogenized in saline, using a Voss homogenizer (initial gut fresh weight: saline, 1:I), the homogenate centrifuged and samples of supernatant fluid also analyzed. By weighing the intestinal sac, empty then filled, both before and after incubation, the serosal fluid transfer and gut fluid uptake were found: the mucosal (total) fluid transfer was also calculated.
INTRODUCTION
Polychlorinated biphenyls (PCBs) are a widespread group of environmental pollutants which are known to have diverse histopathological and physiological effects in mammals and birds. However, the physiological effects of another group of environmental pollutants, polychlorinated terpbenyls (PCTsL have been little studied. The main aim of this work was to investigate the effects of different isomers of PCTs on intestinal absorption of sugars and amino acids and fluid transfer in young adult mice. Studies on the nature of the absorptive process and on the histology of the small intestine were also made. METHODS ,,Inimal~
Male and female mice (strain CD-I), aged between 23 and 4 months, were kept in groups of five in conventional cages with sawdust as bedding in a constant room tempcraturc at 2 2 C ( + I ' C ) and a daylength of 14hr. and given frec access to food (diet 41B) and drinking water. Equal numbers of males and females were used in each experiment.
Substrates and chemical analyses
Different sugars and amino acids were used, each at an initial concentration of 8.0 and 2.0 mmole, respectively. In some experiments, mannose or mannitol (200 mmole each) was also included in the serosal fluid. D-glucose was determined by the Morley et al. (1968) method, D-galactose by the Nelson-Somogyi method (Somogyi, 1952), t,-arginine by Macpherson's (1946) method, and L-histidine by Birt & Hird's (1956) method. All solutions were initially deproteinized in Ba(OH)2-ZnSO 4. Only Analar reagents were used. The final concentration of substrates was expressed as a function of the initial whole small intestinal fresh weight. (The results were not significantly different from those based on a 1.0g fresh weight.) The results were analyzed statistically by paired t-tests, using thc formula for small ,samples (Bailey, 1959).
Inoculation
Samples of PCTs* were dissolved in a known volume of pure corn oil and introduced in single doses directly into the stomach, using a curved hypodermic needle with a beaded tip and a 1 cm 3 syringe. The dose of PCT given to each animal will be stated later. Control (untreated) mice were given corn oil only. In all the experiments the animals were used 1 week after treatment. ]tlle.~litRiJ p r e p a r a t i o n
The conventional everted in vitro intestinal sac preparation was used. Following light anaesthesia in ether, the body-cavity of each animal was opened, the entire small
Histoloyy
The small intestine (n = 5) of treated mice (5(10 mg/kg body weight) was removed, an 8 cm length of the middle region taken, the lumen flushed out with saline, the intestine made into a sac and filled with 0.3 em 3 of alcoholic
* Aroclor 5432, 5442 and 5460; purity 997~,,. Obtained from NEN Chemicals, GmbH. Germany. 43
44
I ) A V I I ) S. MAI)GE
Bouin iixativc. The non-everted sac was left in the fixative for 48 hr, serial sections (7 ~um each) made and stained in eosin and haematoxylin. The sections were compared with those of untreated (control) animals. Particular care was taken to orientate the embedded material so that valid comparisons could be made between inoculated animals and the untreated controls. RESULTS
1. Histoloq)" Few changes were observed between histology of the small intestine of mice given PCI- isomers and that of the controls. The villi were possibly slightly more enlarged and the blood vessels distended, but these results were inconclusive.
2. l)-qhwose absorption and fluid transfer Figure 1 summarizes the absorption of o-glucose 1 week after a single dose of different concentrations of PCTs with chlorine contents of 32, 42 or 600~,. Generally, the higher the dose of P C T given, the lower the D-glucose absorption. Absorption was significantly reduced (P = 0.05-0.001) following 1000. 500 and 250 mg;kg body weight compared to the untreated controls. Although the higher the chlorine content of P C T the greater the absorption, the differences between the PCT isomers were not significant. Following a dose of 50mg/kg, absorption was slightly, but not significantly, lower than that of the controls. Results with 100mg/kg varied: with PCTs having chlorine contents of 32 or 42°~,, absorption was significantly lowered (P = 0.05 for eachl, and with 605~, absorption was not significantly depressed compared to the controls. The D-glucose content of the homogenized tissue [Fig. 1) was generally significantly lower (P = 0.051 following P C T dosages varying from between 1000 18 32
I O00mg 42 60
500rng 32 .'2
60
32
60
250 mg 32 ~2 60
12 i" t-
Z O
~0 er.
32
lOOmg t2 60
Control
UJ (J Z 0
ul 2 Z
";'6
I,
Table 1. Fluid transfer in mouse everted whole small intestine 7 days after a single oral dose of PCT isomers at different concentrations with 32, 42 and 60", chlorine contents Chlorine content ("i,) 1000 mg/kg 32 42 60 500 mg/kg 32 42 60 250 mg/kg 32 42 60 100 mg/kg 32 42 60 50 mg/kg 32 42 60 Control
Initial gut wet wt (g)
Serosal fluid transfer (g)
Gut fluid uptake (g)
1.58 +_ 0.30 1.47 + 0.27 0.23 1.59
1.81 ± 0.21 2.01 ± 0.19 "~ 19 + 0.18
0.76 ± 0.10 0.77 ± 0.11 0.77 + 0.14
1.66 ± 0.29 1.43 ± 0.26 1.60 ± 0.25
1.96 + O.15 2.11 ± 0.19 2.14 +. 0.18
0.80 ± O.13 0.68 _+ 0.16 0.79 _+ 0.11
1.54 ± 0.32 1.53 + 0.30 1.42 ± 0.24
2.16 ± 0.20 2.09 __ 0.19 2.20 ± 0.18
0.78 +__0.14 0.80 ± 0.16 0.77 ± 0.13
1.46 ± 0.32 1.57 ± 0.28 1.39 ± 0.30
1.99 _+ 0.17 2.10 _+ 0.16 2.12 ~ 0.20
0.74 ± 0.11 0.79 ± 0.13 [).75 + 0.14
0.20 0.25 0.25
2.00 ± 0.16 1.96 +_ 0.17 1.99 ± 0.18
0.74 +_ 0.13 0.79 ± 0.13 0.73 + 0.12
0.31
2.09 ± 0.17
0.75 ± 0.14
1.55 1.46 1.59 1.49
± ± + +
D-glucose was used as substrate. Results expressed as absolute values. Means of 10 sacs ± S.E.M.
and 10()mg/kg body weight, but following a dose of 50 mg/kg the D-glucose content was not significantly lower than that of the untreated controls. Table 1 gives the fluid transfer following PCT-treatment at different dosages, using t)-glucose as substrate, and includes the initial small intestinal fresh weight. At PCT dosages varying from 1000 to 5 0 m g / k g body weight, neither the serosal fluid transfer nor the gut fluid uptake were significantly altered compared to the untreated controls. The initial gut wet weight also remained unchanged. Differences between fluid transfer or uptake following PCT-treatment at different dosages were not significant.
3. Intestinal absorption and fluid transfer using other substrates
II MSH
Fig. I. In vitro intestinal absorption of D-glucose using mouse whole everted small intestine 7 days aftcr a single oral dose of PCT isomers with 32. 42 and 60",, chlorine contents. Doses expressed in mg/kg body weight. Each set of histograms reprcsents the final mucosal (M}, scrosal IS) and supernatant of gut tissue homogenate (H) concentrations. Initial concentration of mucosal and serosal fluids 8.0 mmole. Means of 10 cverted sacs. ± S.E.M. as vertical lines. Results of both sexes arc grouped.
Figure 2 and Table 2 show the absorption of m o n o saccharides and amino acids and concomitant total fluid transfer 1 week after single doses of PCTs (500 mg/l
45
Polychlorinated terphenyls and intestinal transport in mice
D-GALACTOSE 2~
32
/,2
ContrOl
32
.D-MALTOSE. |D-GLUCOSEI /,2
v
42
32
~E25
60 Control
+
E12
60
+
I
7
Z O
2-6
Control
+
0
u -J 5
Off I-.7 W U
L-ARGINI NE
L-HISTIDINE
0
O-
z 0
MSH
Fig. 3. Effects of an exogenous energy supply on intestinal absorption of D-glucose 7 days after a single oral dose (500mg/kg body weight) of PUT isomers with different chlorine contents. Initial concentration of D-glucose in mucosal fluid 8.0 mmole and in serosal fluid 8.0 mmole + 200 mmole p-mannose Means of 10 everted sacs, + S.E.M. as vertical lines. Other details as in Fig. 1.
MS/ -/
Fig. 2. Intestinal absorption of different substrates 7 days after a single oral dose (500 mg,&g body weight) of PCT isomers with diffcrent chlorine contents. Initial concentration of sugars 8.0mmolc and that of amino acids 2.0 mmole. Mean of 10 cvertcd sacs, _+S.E.M. as vertical lines. Other details as in Fig. I. Also, differences between substrate absorption or mucosal fluid transfer using the three PCT isomers were not significant. Although the a m o u n t of substrate that accumulated in the tissues during the absorption process depended on the substrate used, the results [=sing PCT-trealed animals and that of the controls were similar. Hey, ever, using l>-maltose, slightly less (P = 0.02-0.05) [>-glucose accumulated in the tissues following PCT-treatment.
4. Effects of an exogenous energy supply The effects of an exogenous energy supply on the absorption of D-glucose and total fluid transfer were investigated by adding D-mannose to the serosal fluid (Smyth, 1971; Madge, 1976b). The results, given in Fig. 3 and Table 3, clearly indicated that under these conditions b o t h D-glucose absorption and total fluid transfer in PCT-treated animals increased. However, both parameters were virtually unchanged compared to untreated controls with an added energy supply [control experiments using mannitol gave similar results to those without mannitol). Differences between D-glucose absorption or total fluid transfer using the different PCTs were also not significant. However. slightly less D-glucose was recovered from the homogenized tissues following doses
of PCT with different chlorine contents compared to the controls (P = 0.05). DISCUSSION
This work investigated the effects of polychlorinated terphenyls on intestinal absorption of different substrates and fluid transfer in adult mice. Although possibly not as widely distributed in the environment as polychlorinated biphenyls (Zitko & Choi, 19711. PCTs may originate from commercial PCB preparations (Anon, 19701 and hence PCTs may be more widespread than hitherto suspected, particularly since industrial applications of PCTs and PCBs are similar. Unlike PCBs (e.g. Madge, 1976a, 1976b), few reports have been published on body accumulation and physiological effects of PCTs in m a m m a l s and birds. For example, PCTs have been detected in the Table 3. Effects of an exogenous energy supply (D-mannose in serosal fluid) and fluid transfer in mouse everted small intestine 7 days after a single oral dose (500 mg/kg body weight) of PCT isomers with different per cent chlorine contents, using [)-glucose as substrate Chlorine content (%)
Initial gut wet wt (g)
Serosal fluid transfer (g)
Gut fluid uptake (g)
32 42 60
1.32 _+ 0.33 1.31 _+ 0.33 1.48 _____0.27
2.42 _+ 0.16 2.49 -L 0.16 2.40 _+ 0.19
0.88 + 0.12 0.85 --t-__0.13 0.86 -+ 0.11
Control
1.41 _+ 0.30
2.46 _+ 0.18
0.86 + 0.12
Means of 10 sacs _+ S.E.M.
Table 2. Mucosal fluid transfer in mouse everted whole small intestine seven days after a single oral dose (500 mg/kg body' weight} of PCT isomers with different per cent chlorine contents, using different substrates Subs(rate l)-galactose p-maltose L-arginine L-histidine
32". 1.44 + 0.25 1.41 _+ (I.21 1.39 + 0.23 1.34 -+ 0.26
Initial gut weight (g) 42% 60% 1.44 + 0.30 1.47 + 0.20 1.50 _+ 0.3(I 1.40 -4-__0.23
Results expressed as absolute values. Means of 10 sacs + S.E.M.
1.36 + 0.24 1.49 _+ 0.26 1.39 __-4-0.21 1.35 -+ 0.20
Control
32%
1.49 _+ 0.27 1.40 _+ 0.26 1.42 ___+(I.29 1.47 -+ 0.27
1.11 +0.13 2.85_+(I.09 (I.77 + 0.08 0.89 __+0.10
Mucosal fluid transfer (g) 42% 60";, 0.99__+0.11 2.89-t-0.13 0.80 _+ 0.07 (I.85 _+ 0.10
Control
0.97__0.13 1.10__+0.14 2.91 __-t-0.11 2.87-+0.12 (I.79 -+ 0.07 0.75 _+ 0.08 0.88 _+ 0.09 0.81 t- 0.09
46
DAVID S. MADGE
eggs and subcutaneous fatty tissue of herring gulls {Larus aryentatus) from Canada (Zitko et al., 1972). Present results indicated that following single doses of three PCT isomers having chlorine contents of 32, 42 and 60041 D-glucose absorption was depressed after 1 week at dosages varying from 1000 to 250 mg/kg body weight compared to untreated controls; at a dose of 100 mg/kg the results varied, and at a dose of 50mg/kg absorption was unaltered. Total fluid transfer remained unchanged at all dosages of PCTs. However, further evidence suggested that malabsorption of D-glucose might have resulted from an effect other than malfunction at the microvillous (mucosal) border. When an exogenous energy supply, o-mannose, which is known to diffuse through the intestinal tissues, was added to the serosal fluid so providing the absorptive cells with an added source of energy tSmyth, 1971), differences in D-glucose absorption were not found between PCT-treated animals and the untreated controls with added energy supply. These results suggested that depressed D-glucose absorption in PCT-treated mice without exogenous energy in the previous experiments probably resulted from malfunction of the overall metabolism of the absorptive cells which indirectly affected the absorption process. Hence, D-glucose, which was both transferred and metabolized, was metabolized to a greater extent in PCT-treated animals during intracellular transfer, resulting in an apparent decrease in overall o-glucose absorption. The fact that the final homogenate concentrations of D-glucose in PCT-treated mice were lower than in those of the controls also indicated that o-glucose was metabolized during the absorption process. Generally, therefore, PCTs appeared to affect mainly the inherent endogenous energy of the absorptive cells and not the transport of D-glucose into the cells. This conclusion was substantiated by using other substrates (D-galactose, L-arginine and L-histidine) which were transferred but not metabolized, when substrate absorption and fluid transfer in PCTtreated animals remained unaffected. Finally, the absorption of D-glucose following hydrolysis of D-maltose in both PCT-treated and control mice was alike, suggesting that the sodium-independent carrier (Caspary, 1976) responsible for D-glucose transport following hydrolysis of the disaccharide remained unimpaired by PCTs, like the sodium-dependent carrier responsible for o-glucose transport when only the monosaccharide was umd as substrate (Crane. 1965). The effects of PCT isomers on D-glucose absorption and those of unpurified isomers of PCBs (at acute doses) were alike (Madge. 1976a). Moreover, apparent malabsorption of D-glucose was also obtained after
Phenoclor DP6 treatment, a PCB compound containing traces of toxic pentodichlorobenzofuran, but not after Pyralene 1500 or 3010 treatment, which arc purified PCB compounds (Madge, 1976b), suggesting that this toxic impurity was partly or mainly responsible for the malabsorptive effect. Thus, experiments on intestinal absorption and fluid transfer using PCBs and PCTs have suggested that isomers of both compounds affected mainly the metabolism of the absorptive cells and only indirectly their absorptive process. Acknowledqement.s -I am grateful tu Messrs. Brian l)odson and John Haines for their technical assistance and Dr. A. M. Scofield for criticizing the manuscript.
REFERENCI.k~; ANON. (1972) Analahs Research Notes 12, 13. BAILEY N. T. J. (1959) Statistical Method.s in Biolo~g.v. English Universities Press. BIRI L. M. & HIRD J. J. R. (1956) The uptake of amino acids by carrot slices. Biochem. J. 64, 305 311. CASPARY W. F. (1976) Ionic dependence of glucose transport from disaccharides. Intestinal hm Transport (Edited by ROBINSON, J. W. L.), pp. 153 154. MTP, Lancaster. CRA,~E R. K. (1965} Sodium-dependent transport in the intestine and other animal tissues. Fedn Proc. I:edn Am. Socs exp. Biol. 24, 1000-1006. MACPHERSON H. T. (1946) The basic amino-acid content of proteins. Biochem. J. 40, 470- 481. MAt~E D. S. (1976a) Polychlorinated biphen.vls and intestinal absorption of D-glucose in mice. Go~. Pharmac. 7. 45-48. MAI×;E D. S. (1976b) Polychlorinatcd hiphcnyls (phcnoclor and pyralene) and intestinal translx~rt of hexoses and amino acids in mice. Gen. Pharmac. 7. 249 254. MORLEY G.. DAWSOYA. & MARKS V. (19681 Manual and autoanalyzer methods for measuring blood glucose using guaiacum and glucose oxidase. Proc. A,ss. din. Biochem. 5, 42 45. SMYTH D. H. 11971) Sodium-hexose interactions. Phil. Trans. R. Soc. Set. B. 262, 121. 130. SOMOGW M. (1952) Notes on sugar determination, d. biol. Chem. 195, 19.23. Z1TKO V. & CHOI P. M. K. [1971~ PCB and other industrial halogenated hydrocarbons in the environment. Fi.sh. Re.s. Bd Can. Tech. Rep. No. 272. ZI'IKO V.. HUTZINGERO.. JAMIE~)N W. D. & ('llOl P. M. K. 09721 Polychlorinated terphcnyls in the enxironment. Bull. Ent. Contain. Toxic. 7, 200 201. NOTE ADDED IN
PROOF
PCTs have recently been detected m xsildlifc species from Europe, in human fat from Japan and in food packaging material. ZlrKo V. 11975) Potentiall.~ persistent industrial organic chemicals other than PCB. l-colo~lical Toxicolo.qy Research (Edited b~ Mcl,,,,rYRt A. D. & Mit I,s C. F.), pp. 197-206. Plenum Press. New York
POLYCHLORINATED TERPHENYLS A N D INTESTINAL TRANSPORT IN MICE DAVID S. MADGE Unit of Applied Zoology, Wye College (University of London), Ashford. Kent TN25 5AH, England (Received 10 October 1976)
Abstract I. Investigations were made on in vitro intestinal absorption of different substrates and fluid transfer in mice following single oral doses of isomers of polychlorinated terphenyls {PCTs). 2. Depending on the dose, D-glucose absorption, but not fluid transfer, was depressed following PC'T-treatment, as was the D-glucose content of tissue homogenates. Malabsorption was generally unrelated to the chlorine content of the PCTs. However, absorption of D-galactose, L-arginine and I,-histidinc. including fluid transfer, remained unaltered. 3. Malabsorption of D-glucose probably resulted from intracellular metabolic effects of PCTs and not malfunction of the D-glucose carrier at the mucosal membrane. 4. Thc histology of the small intestine was unchanged, as was the wet intestinal weight. 5. Absorption experiments using PCTs were compared with previous work on PCBs, and suggested that both malabsorptive effects were similar.
intestine quickly removed, its lumen flushed out with 0.9",, saline solution, and the intestine turned inside out using a slender steel rod. One end of the intestine was ligatured, the intestine filled with 1.25 cm 3 of oxygenated KrebsRinger bicarbonate solution containing the dissolved substrate (serosal fluid), and the other end then ligatured Each sac was put into a 150cm a Erlenmeyer flask containing 25 cm 3 of the same test solution and substrate (mucosal fluid), and the air inside the flask replaced with a mixture of 95% 0 2 + 5°0 CO2. The whole procedure took about 8 min. The flask was then shaken (80 oscillations/min and 2 cm amplitude) in a water-bath at 37C for 1 hr. Following incubation, samples of the serosal and mucosal fluid were analyzed for the appropriate test substrate. The empty sac was washed in saline and the tissue then homogenized in saline, using a Voss homogenizer (initial gut fresh weight: saline, 1:I), the homogenate centrifuged and samples of supernatant fluid also analyzed. By weighing the intestinal sac, empty then filled, both before and after incubation, the serosal fluid transfer and gut fluid uptake were found: the mucosal (total) fluid transfer was also calculated.
INTRODUCTION
Polychlorinated biphenyls (PCBs) are a widespread group of environmental pollutants which are known to have diverse histopathological and physiological effects in mammals and birds. However, the physiological effects of another group of environmental pollutants, polychlorinated terpbenyls (PCTsL have been little studied. The main aim of this work was to investigate the effects of different isomers of PCTs on intestinal absorption of sugars and amino acids and fluid transfer in young adult mice. Studies on the nature of the absorptive process and on the histology of the small intestine were also made. METHODS ,,Inimal~
Male and female mice (strain CD-I), aged between 23 and 4 months, were kept in groups of five in conventional cages with sawdust as bedding in a constant room tempcraturc at 2 2 C ( + I ' C ) and a daylength of 14hr. and given frec access to food (diet 41B) and drinking water. Equal numbers of males and females were used in each experiment.
Substrates and chemical analyses
Different sugars and amino acids were used, each at an initial concentration of 8.0 and 2.0 mmole, respectively. In some experiments, mannose or mannitol (200 mmole each) was also included in the serosal fluid. D-glucose was determined by the Morley et al. (1968) method, D-galactose by the Nelson-Somogyi method (Somogyi, 1952), t,-arginine by Macpherson's (1946) method, and L-histidine by Birt & Hird's (1956) method. All solutions were initially deproteinized in Ba(OH)2-ZnSO 4. Only Analar reagents were used. The final concentration of substrates was expressed as a function of the initial whole small intestinal fresh weight. (The results were not significantly different from those based on a 1.0g fresh weight.) The results were analyzed statistically by paired t-tests, using thc formula for small ,samples (Bailey, 1959).
Inoculation
Samples of PCTs* were dissolved in a known volume of pure corn oil and introduced in single doses directly into the stomach, using a curved hypodermic needle with a beaded tip and a 1 cm 3 syringe. The dose of PCT given to each animal will be stated later. Control (untreated) mice were given corn oil only. In all the experiments the animals were used 1 week after treatment. ]tlle.~litRiJ p r e p a r a t i o n
The conventional everted in vitro intestinal sac preparation was used. Following light anaesthesia in ether, the body-cavity of each animal was opened, the entire small
Histoloyy
The small intestine (n = 5) of treated mice (5(10 mg/kg body weight) was removed, an 8 cm length of the middle region taken, the lumen flushed out with saline, the intestine made into a sac and filled with 0.3 em 3 of alcoholic
* Aroclor 5432, 5442 and 5460; purity 997~,,. Obtained from NEN Chemicals, GmbH. Germany. 43
44
I ) A V I I ) S. MAI)GE
Bouin iixativc. The non-everted sac was left in the fixative for 48 hr, serial sections (7 ~um each) made and stained in eosin and haematoxylin. The sections were compared with those of untreated (control) animals. Particular care was taken to orientate the embedded material so that valid comparisons could be made between inoculated animals and the untreated controls. RESULTS
1. Histoloq)" Few changes were observed between histology of the small intestine of mice given PCI- isomers and that of the controls. The villi were possibly slightly more enlarged and the blood vessels distended, but these results were inconclusive.
2. l)-qhwose absorption and fluid transfer Figure 1 summarizes the absorption of o-glucose 1 week after a single dose of different concentrations of PCTs with chlorine contents of 32, 42 or 600~,. Generally, the higher the dose of P C T given, the lower the D-glucose absorption. Absorption was significantly reduced (P = 0.05-0.001) following 1000. 500 and 250 mg;kg body weight compared to the untreated controls. Although the higher the chlorine content of P C T the greater the absorption, the differences between the PCT isomers were not significant. Following a dose of 50mg/kg, absorption was slightly, but not significantly, lower than that of the controls. Results with 100mg/kg varied: with PCTs having chlorine contents of 32 or 42°~,, absorption was significantly lowered (P = 0.05 for eachl, and with 605~, absorption was not significantly depressed compared to the controls. The D-glucose content of the homogenized tissue [Fig. 1) was generally significantly lower (P = 0.051 following P C T dosages varying from between 1000 18 32
I O00mg 42 60
500rng 32 .'2
60
32
60
250 mg 32 ~2 60
12 i" t-
Z O
~0 er.
32
lOOmg t2 60
Control
UJ (J Z 0
ul 2 Z
";'6
I,
Table 1. Fluid transfer in mouse everted whole small intestine 7 days after a single oral dose of PCT isomers at different concentrations with 32, 42 and 60", chlorine contents Chlorine content ("i,) 1000 mg/kg 32 42 60 500 mg/kg 32 42 60 250 mg/kg 32 42 60 100 mg/kg 32 42 60 50 mg/kg 32 42 60 Control
Initial gut wet wt (g)
Serosal fluid transfer (g)
Gut fluid uptake (g)
1.58 +_ 0.30 1.47 + 0.27 0.23 1.59
1.81 ± 0.21 2.01 ± 0.19 "~ 19 + 0.18
0.76 ± 0.10 0.77 ± 0.11 0.77 + 0.14
1.66 ± 0.29 1.43 ± 0.26 1.60 ± 0.25
1.96 + O.15 2.11 ± 0.19 2.14 +. 0.18
0.80 ± O.13 0.68 _+ 0.16 0.79 _+ 0.11
1.54 ± 0.32 1.53 + 0.30 1.42 ± 0.24
2.16 ± 0.20 2.09 __ 0.19 2.20 ± 0.18
0.78 +__0.14 0.80 ± 0.16 0.77 ± 0.13
1.46 ± 0.32 1.57 ± 0.28 1.39 ± 0.30
1.99 _+ 0.17 2.10 _+ 0.16 2.12 ~ 0.20
0.74 ± 0.11 0.79 ± 0.13 [).75 + 0.14
0.20 0.25 0.25
2.00 ± 0.16 1.96 +_ 0.17 1.99 ± 0.18
0.74 +_ 0.13 0.79 ± 0.13 0.73 + 0.12
0.31
2.09 ± 0.17
0.75 ± 0.14
1.55 1.46 1.59 1.49
± ± + +
D-glucose was used as substrate. Results expressed as absolute values. Means of 10 sacs ± S.E.M.
and 10()mg/kg body weight, but following a dose of 50 mg/kg the D-glucose content was not significantly lower than that of the untreated controls. Table 1 gives the fluid transfer following PCT-treatment at different dosages, using t)-glucose as substrate, and includes the initial small intestinal fresh weight. At PCT dosages varying from 1000 to 5 0 m g / k g body weight, neither the serosal fluid transfer nor the gut fluid uptake were significantly altered compared to the untreated controls. The initial gut wet weight also remained unchanged. Differences between fluid transfer or uptake following PCT-treatment at different dosages were not significant.
3. Intestinal absorption and fluid transfer using other substrates
II MSH
Fig. I. In vitro intestinal absorption of D-glucose using mouse whole everted small intestine 7 days aftcr a single oral dose of PCT isomers with 32. 42 and 60",, chlorine contents. Doses expressed in mg/kg body weight. Each set of histograms reprcsents the final mucosal (M}, scrosal IS) and supernatant of gut tissue homogenate (H) concentrations. Initial concentration of mucosal and serosal fluids 8.0 mmole. Means of 10 cverted sacs. ± S.E.M. as vertical lines. Results of both sexes arc grouped.
Figure 2 and Table 2 show the absorption of m o n o saccharides and amino acids and concomitant total fluid transfer 1 week after single doses of PCTs (500 mg/l
45
Polychlorinated terphenyls and intestinal transport in mice
D-GALACTOSE 2~
32
/,2
ContrOl
32
.D-MALTOSE. |D-GLUCOSEI /,2
v
42
32
~E25
60 Control
+
E12
60
+
I
7
Z O
2-6
Control
+
0
u -J 5
Off I-.7 W U
L-ARGINI NE
L-HISTIDINE
0
O-
z 0
MSH
Fig. 3. Effects of an exogenous energy supply on intestinal absorption of D-glucose 7 days after a single oral dose (500mg/kg body weight) of PUT isomers with different chlorine contents. Initial concentration of D-glucose in mucosal fluid 8.0 mmole and in serosal fluid 8.0 mmole + 200 mmole p-mannose Means of 10 everted sacs, + S.E.M. as vertical lines. Other details as in Fig. 1.
MS/ -/
Fig. 2. Intestinal absorption of different substrates 7 days after a single oral dose (500 mg,&g body weight) of PCT isomers with diffcrent chlorine contents. Initial concentration of sugars 8.0mmolc and that of amino acids 2.0 mmole. Mean of 10 cvertcd sacs, _+S.E.M. as vertical lines. Other details as in Fig. I. Also, differences between substrate absorption or mucosal fluid transfer using the three PCT isomers were not significant. Although the a m o u n t of substrate that accumulated in the tissues during the absorption process depended on the substrate used, the results [=sing PCT-trealed animals and that of the controls were similar. Hey, ever, using l>-maltose, slightly less (P = 0.02-0.05) [>-glucose accumulated in the tissues following PCT-treatment.
4. Effects of an exogenous energy supply The effects of an exogenous energy supply on the absorption of D-glucose and total fluid transfer were investigated by adding D-mannose to the serosal fluid (Smyth, 1971; Madge, 1976b). The results, given in Fig. 3 and Table 3, clearly indicated that under these conditions b o t h D-glucose absorption and total fluid transfer in PCT-treated animals increased. However, both parameters were virtually unchanged compared to untreated controls with an added energy supply [control experiments using mannitol gave similar results to those without mannitol). Differences between D-glucose absorption or total fluid transfer using the different PCTs were also not significant. However. slightly less D-glucose was recovered from the homogenized tissues following doses
of PCT with different chlorine contents compared to the controls (P = 0.05). DISCUSSION
This work investigated the effects of polychlorinated terphenyls on intestinal absorption of different substrates and fluid transfer in adult mice. Although possibly not as widely distributed in the environment as polychlorinated biphenyls (Zitko & Choi, 19711. PCTs may originate from commercial PCB preparations (Anon, 19701 and hence PCTs may be more widespread than hitherto suspected, particularly since industrial applications of PCTs and PCBs are similar. Unlike PCBs (e.g. Madge, 1976a, 1976b), few reports have been published on body accumulation and physiological effects of PCTs in m a m m a l s and birds. For example, PCTs have been detected in the Table 3. Effects of an exogenous energy supply (D-mannose in serosal fluid) and fluid transfer in mouse everted small intestine 7 days after a single oral dose (500 mg/kg body weight) of PCT isomers with different per cent chlorine contents, using [)-glucose as substrate Chlorine content (%)
Initial gut wet wt (g)
Serosal fluid transfer (g)
Gut fluid uptake (g)
32 42 60
1.32 _+ 0.33 1.31 _+ 0.33 1.48 _____0.27
2.42 _+ 0.16 2.49 -L 0.16 2.40 _+ 0.19
0.88 + 0.12 0.85 --t-__0.13 0.86 -+ 0.11
Control
1.41 _+ 0.30
2.46 _+ 0.18
0.86 + 0.12
Means of 10 sacs _+ S.E.M.
Table 2. Mucosal fluid transfer in mouse everted whole small intestine seven days after a single oral dose (500 mg/kg body' weight} of PCT isomers with different per cent chlorine contents, using different substrates Subs(rate l)-galactose p-maltose L-arginine L-histidine
32". 1.44 + 0.25 1.41 _+ (I.21 1.39 + 0.23 1.34 -+ 0.26
Initial gut weight (g) 42% 60% 1.44 + 0.30 1.47 + 0.20 1.50 _+ 0.3(I 1.40 -4-__0.23
Results expressed as absolute values. Means of 10 sacs + S.E.M.
1.36 + 0.24 1.49 _+ 0.26 1.39 __-4-0.21 1.35 -+ 0.20
Control
32%
1.49 _+ 0.27 1.40 _+ 0.26 1.42 ___+(I.29 1.47 -+ 0.27
1.11 +0.13 2.85_+(I.09 (I.77 + 0.08 0.89 __+0.10
Mucosal fluid transfer (g) 42% 60";, 0.99__+0.11 2.89-t-0.13 0.80 _+ 0.07 (I.85 _+ 0.10
Control
0.97__0.13 1.10__+0.14 2.91 __-t-0.11 2.87-+0.12 (I.79 -+ 0.07 0.75 _+ 0.08 0.88 _+ 0.09 0.81 t- 0.09
46
DAVID S. MADGE
eggs and subcutaneous fatty tissue of herring gulls {Larus aryentatus) from Canada (Zitko et al., 1972). Present results indicated that following single doses of three PCT isomers having chlorine contents of 32, 42 and 60041 D-glucose absorption was depressed after 1 week at dosages varying from 1000 to 250 mg/kg body weight compared to untreated controls; at a dose of 100 mg/kg the results varied, and at a dose of 50mg/kg absorption was unaltered. Total fluid transfer remained unchanged at all dosages of PCTs. However, further evidence suggested that malabsorption of D-glucose might have resulted from an effect other than malfunction at the microvillous (mucosal) border. When an exogenous energy supply, o-mannose, which is known to diffuse through the intestinal tissues, was added to the serosal fluid so providing the absorptive cells with an added source of energy tSmyth, 1971), differences in D-glucose absorption were not found between PCT-treated animals and the untreated controls with added energy supply. These results suggested that depressed D-glucose absorption in PCT-treated mice without exogenous energy in the previous experiments probably resulted from malfunction of the overall metabolism of the absorptive cells which indirectly affected the absorption process. Hence, D-glucose, which was both transferred and metabolized, was metabolized to a greater extent in PCT-treated animals during intracellular transfer, resulting in an apparent decrease in overall o-glucose absorption. The fact that the final homogenate concentrations of D-glucose in PCT-treated mice were lower than in those of the controls also indicated that o-glucose was metabolized during the absorption process. Generally, therefore, PCTs appeared to affect mainly the inherent endogenous energy of the absorptive cells and not the transport of D-glucose into the cells. This conclusion was substantiated by using other substrates (D-galactose, L-arginine and L-histidine) which were transferred but not metabolized, when substrate absorption and fluid transfer in PCTtreated animals remained unaffected. Finally, the absorption of D-glucose following hydrolysis of D-maltose in both PCT-treated and control mice was alike, suggesting that the sodium-independent carrier (Caspary, 1976) responsible for D-glucose transport following hydrolysis of the disaccharide remained unimpaired by PCTs, like the sodium-dependent carrier responsible for o-glucose transport when only the monosaccharide was umd as substrate (Crane. 1965). The effects of PCT isomers on D-glucose absorption and those of unpurified isomers of PCBs (at acute doses) were alike (Madge. 1976a). Moreover, apparent malabsorption of D-glucose was also obtained after
Phenoclor DP6 treatment, a PCB compound containing traces of toxic pentodichlorobenzofuran, but not after Pyralene 1500 or 3010 treatment, which arc purified PCB compounds (Madge, 1976b), suggesting that this toxic impurity was partly or mainly responsible for the malabsorptive effect. Thus, experiments on intestinal absorption and fluid transfer using PCBs and PCTs have suggested that isomers of both compounds affected mainly the metabolism of the absorptive cells and only indirectly their absorptive process. Acknowledqement.s -I am grateful tu Messrs. Brian l)odson and John Haines for their technical assistance and Dr. A. M. Scofield for criticizing the manuscript.
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PROOF
PCTs have recently been detected m xsildlifc species from Europe, in human fat from Japan and in food packaging material. ZlrKo V. 11975) Potentiall.~ persistent industrial organic chemicals other than PCB. l-colo~lical Toxicolo.qy Research (Edited b~ Mcl,,,,rYRt A. D. & Mit I,s C. F.), pp. 197-206. Plenum Press. New York