Action of dibenzofurans and naphthalenes on intestinal solute transport and fluid transfer in mice

Gtm. Pharmuc.. t.ol 9. pp 361 to 367 Pt.r~lamOll Pre~ Lid 197g Prortcd m Great Britul.

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ACTION OF DIBENZOFURANS AND NAPHTHALENES ON INTESTINAL SOLUTE T R A N S P O R T A N D F L U I D T R A N S F E R IN MICE DAVID S. MADGE Unit of Applied Zoology. Wye College (University of London), Near Ashford, Kent TN25 5AH, U.K (Receired 9 February 19781 Abstract--I. Investigations were made on the effects of a single, oral dose of purified chlorinated dibenzofuran and non-chlorinated dibenzofuran and polychlorinated naphthalenes on in citro absorption of various solutes and concomitant fluid transfer in the mouse everted small intestine. 2. Absorption of D-glucose decreased 7 days after treatment with each of these chemicals, as did the final D-glucose content of tissue homogenates. D-glucose absorption following membrane hydrolysis of D-maltose was unaltered. D-glucose malabsorption was abolished by an exogenous energy supply (D-mannose). Intestinal absorption of o-galactose and several amino acids was unchanged. Fluid transfer remained unaltered with each of the solutes. 3. Lowered D-glucose absorption in the experimental animals probably resulted from increased intracellular metabolic effects following absorption and not from impaired function of the ~glucose carrier at the brush border membrane. 4. The apparent'malabsorptive effects of D-glucose was both time-dependent and dose-dependent. Dibenzofurans were effective at considerably lower doses compared with naphthalenes. 5. Apparent malabsorption was unrelated to the chlorine content of the chemicals. 6. Histological changes of the small intestine were small and inconsistent: the fresh intestinal weight remained unchanged. 7. The apparent malabsorptive effects of D-glucose using commercial polychlorinated biphcnyls and polychlorinated terphenyls were probably caused by traces of toxic chemicals, particularly dibenzofuran. in these compounds.

MATERIALS AND METHODS

INTRODUCTION

Commercial polychlorinated biphenyl (PCB) and polychlorinated terphenyl (PCT) c o m p o u n d s are widely dispersed environmental pollutants which can accumulate in animal tissues and cause diverse histopathological and biochemical effects. Traces of toxic impurities in these c o m p o u n d s appear to be mainly responsible for producing such effects, particularly chlorinated dibenzofurans and to a lesser extent polychlorinated naphthalenes (Vos, 1972: Fishbein. 19741. Previous work on PCBs (Madge, 1976a, b) and PCTs (Madge, 1977a) in mice showed that in vitro intestinal absorption of t)-glucose apparently decreased following a single, oral dose of these compounds while intestinal fluid transfer was unaltered. In contrast, intestinal absorption of t)-galactose and several a m i n o acids remained unchanged, as did fluid transfer. Further experiments suggested that the apparent malabsorption of o-glucose probably resulted from increased metabolism of the solute in the affected tissues during the experiment and not from malfunction of the D-glucose transfer mechanism at the brush border. The main purpose of this work was to investigate the effects of chlorinated and non-chlorinated dibenzofurans and polychlorinated naphthalenes, which are toxic impurities in PCBs and PCTs. on intestinal absorption of monosaccharides and amino acids and fluid transfer in mice. The absorptive mechanism and the histology of the small intestine were also studied.

Animals Mice (inbred strain CD-I) of both sexes were used. aged between 2½ and 4½ months. The animals were kept in groups of 4-5 in standard animal cages, at a constant temperature of 22 _+ 2~C and 14 hr artificial light, using sawdust as bedding. The animals were given an unrestricted supply of pelletted food (Oxoid 41B) and drinking water. Inoculation Polychlorinated dibenzofuran (I.2.3.4,5.6.7.8-octachlorodibenzofuran), of 997J,~ purity, and non-chlorinated dibenzofuran, of 98'~, purity, were both obtained from N E N Chemicals. GmbH. Germany (lots RCS 070 and RCS 071 respectively). Polychlorinated naphthalenes, of 99°,; purity. obtained from NEN Chemicals (Halowax kit, lot RCS 0761 were also used. having the following chlorine contents: 22 (Halowax No. 10311, 26 (I0001, 50 (10OIL 52 (10991, 56 (1013), 62 (10141, and 70 (1051) per cent. The concentration of the chemicals used varied in different experiments and will be given later. Groups of 8 or 10 animals (equal number of each sex) were.each given a single, oral dose of the chemical dissolved in 0.3 cm 3 of pure corn oil, using a I cm 3 hypodermic syringe and needle with a curved, beaded tip. and in most experiments the animals were used 7 days later. Control animals were each given 0.3 cm 3 of pure corn oil only. Histology Groups of four mice were each given a single dose of polychlorinated dibenzofuran, non-chlorinated dibenzofuran (2.0mgkg -1 body wt each) and polychlorinated naphthalenes having 22?0 and 70",, chlorine content 361

362

D A V I D S. M A I ) G I

(I.0g kg ~ bod.~ wt each). Sc,,cn da)s later the small intestine of each animal was remo,,ed, cvertcd, filled with alcoholic Bouin. and the sacs wcrc left in the fixative for 3 da.',s. Three l-cm portions of the mid-intestine of each animal were then removed, the tissues of different animals in each group pooled and three pieces from each group taken at random, dehydrated, processed, sectioned at 7 l~m and stained with eosin and celestin blue. The prepared sections of the different treatments were compared with each other and with the untreated evcrted intestine.

lnt¢.~tinal .~ac preparation The everted m mtro intestinal sac method was used to determine intestinal absorption of solutes and fluid transfer, as in pre~,ious studies [Madgc. 1976a. b: 1977a1. The animal was lightly anaesthetized with ether, the bodycavity opened and the entire small intestine quickly removed. The intestinal lumen was flushed with O9",, saline solution and the intestine turned inside out using a thin metal rod. The cverted intestinal surface was cleaned by dipping the intestine in saline solution and then lightly blotting its surface. One end of the intestine was ligaturcd and the sac filled with 1.25 cm 3 of oxygenated Krebs saline bicarbonate solution containing the dissolved solute (serosal solution) using a I cm 3 hypodermic syringe and a straight, blunt-ended needle. The other end was ligatured. the preparation tested for leaks, the sac dropped into a 150cm 3 Erlenmeyer flask containing 25.0cm ~ of the same solution [mucosal fluid), and the flask gassed for 2 3 min in a mixturc of 95°o oxygen and 5°. carbon dioxide. The flask was placed in a water-bath at 37'C and shaken (70 oscillations min- ~, 2 cm amplitude) for 60 min. The sac was then removed and the serosal fluid recovered. The sac was dipped in saline solution and the lumen flushed in saline, the preparation lightly blotted and the intestinal tissue homogenized in saline (ratio tissue: saline, 1 : I I and the supernatant recovered. The solute concentration in samples of mucosal and scrosal fluids and in the tissue supernatant was then found b.~ chemical analysis. By weighing the intestinal sac before and after incubation, empty and tilled, the serosal tluid transfer and gut fluid uptake were also found.

and slightly distended. The tissues of the treated animals, particularly at high dosages, were more fragile than those o f the c o n t r o l s and thus m o r e difficult to evert. The fresh small intestinal weight of the treated animals and that of thc c o n t r o l s was alike. M o s t of the a n i m a l s survived the treatment.

Intestinal absorption and fluid transJer Since differences between male and female a n i m a l s wcrc not significant, the data have been grouped. (1) Polychlorinated dihenz~i:ran (PCDF)

E~g~'ct.s oJd!fferent do.ses. Figure I s h o w s the effects o f single d o ~ s of P C D F varying from 0.25 to 1 0 . 0 m g k g -~ b o d y wt after 7 days on intestinal transfer o f [~glucose. T h e higher the dose the greater the m a l a b s o r p t i o n . A b s o r p t i o n wa,; not significantly lower than in the c o n t r o l s following a dose o f either 0.25 or 0 . 5 0 m g k g -] body wt. After a dose of 1 . 0 m g k g ~ body wt a b s o r p t i o n was signiticantly reduced (P = 0.05) which became intensified after a dose of 2.0. 5.0 or 10.0 mg k g - ~ (P = 0.001 each). T h e minimal effective single dose of this chemical giving consistently m a r k e d m a l a b s o r p t i o n . 2.0 m g kg " z body wt, was c h o ~ n for s u b s e q u e n t experiments. T h e tissue c o n c e n t r a t i o n of D-glucose following incubation generally decreased the higher the dosage. For example, it was 1.6 + 0.07, 0.91 + 0.09, 0.41 + 0.05 a n d 0.36 + 0.04 m - m o l a r following doses o f 0.25. 1.0, 2.0, and 1 0 . 0 m g k g -~ body wt respectively: for the controls it was 1.8 + 0.08 m - m o l a r (P = 0.05 for doses of 1.O 10.0 m g kg - ~). Thc s e r o ~ l fluid transfer and gut fluid uptake following different doscs of P C D F were not significantl? different c o m p a r e d to the c o n t r o l s {detailed results 2015

Solute.s and analy.sc.~ Two monosaccharides, o-glucose and o-galactose, each at an initial concentration of 8.0 m-molar, and four amino acids. L-argininc, L-histidine, t.-ornithine and L-proline. each at an initial concentration of 2.0 m-molar, wcrc used in both the mucosal and serosal fluids in separate experiments. One disaccharidc, D-maltose. at an initial concentration o f f . 0 m-molar, was also used. In some experiments. 200 m-molar of t)-mannose was added to D-glucose in the serosal fluid. o-Glucose was ~:nalyzed b~, the guaiacum-glucose oxidase method (Morle.,, et al., 1968). o-galactose by the Nelson-Somogyi method (Somogyi, 1952), t_-arginine by Macpherson's (1946) method. L-histidine by Birt & Hird's (1956) method, and L-ornithine and t.-proline by Chinard's (19521 method. All the samples were initially deproteinized in Ba(OH)2-ZnS04. Only Analar reagents were used. The results were analysed statisticall~ by t-tests, using N-I weighting (Bailey. 1959).

10 E c o

c u c

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Histoloyy C o m p a r e d with the controls, the histology o f the mid-intestine u n d e r the light m i c r o s c o p e in all treated a n i m a l s s h o w e d only m i n i m a l changes. T h e surface o f the villi a p p e a r e d n o r m a l , a l t h o u g h the villi were s o m e t i m e s slightly swollen. The lamina p r o p r i a a n d s u b m u c o s a l tissues were occasionally o e d a m o t o u s

0-25

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20--

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S C Fig. I. Intestinal absorption of tr-glucose 7 days after a single dose (mg kg- ~ body wtJ of chlorinated dibenzofuran at different concentrations. C: untreated controls. Each paired histogram represents the final mucosal (M) and serosal (SJ concentration. Initial concentration of mucosal and serosal fluid: 8.0m-molar. Means of 8 intestinal sacs (animals); + S.E.M. as vertical lines. Results of both sexes are grouped.

Action of dibenzofurans and naphthalenes

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Fig. 2. Intestinal absorption of o-glucose at varying periods of 1-28 days after a singledose of chlorinated dibenzofuran at a concentration of 2.0 mg kg-1 body wt. Other details as in Fig. I. not given). The serosal fluid transfer after P C D F treatment at all dosages averaged 2.03 4- 0.16 g; that of the controls was 1.99 4- 0.15 g. The gut fluid uptake averaged 0.72 + 0.14 g and 0.68 + 0.09 g respectively. Effects of time. Figure 2 summarizes the effect of the single dose of 2.0 mg k g - ' body wt P C D F after intervals varying from I to 28 days on intestinal absorption of D-glucose. Absorption was slightly reduced after 1 day's treatment (result not significant). which progressively decreased after 3 days (P = 0.02-0.05) and 5 days (P = 0.05). Absorption remained consistently low between 7 and 14 days (P = 0.001l, but tended to increase after 21 and 28

5"

Arglnme

363

days although it remained significantly lower than in the controls (P = 0.01 and 0.05 respectively). The final D-glucose tissue content after 1, 3, 5 and 7 days averaged 1.10 + 0.07. 0.57 ___0.04, 0.40 + 0.05 and 0.41 + 0.05m-molar respectively. It thereafter remained consistently low (0.34 - 0.38 m-molar) although it increased slightly (0.46 + 0.06 m-molar) after 28 days. That of the controls was 1.8 + 0.08 m-molar. Both the serosal fluid transfer and gut fluid uptake in mice given single doses of P C D F at different intervals were close. The average serosal fluid transfer in the treated animals was 1.97 + 0.18 g and that of the controls 1.99 + 0.23g; the gut fluid uptake was 0.79 + 0.17g and 0.68 + 0.09 g respectively. Absorption of other solutes and fluid transfer. Figure 3 compares the intestinal absorption of D-galactose and four different amino acids in PCDF-treated animals with that of the untreated controls; the final solute tissue accumulation is also included. Generally. differences between the treated mice and those of the controls were small and not signifieant. Table 1 summarizes the serosal fluid transfer and gut fluid uptake in the different experiments. Again, there were no significant differences between the experimental animals and the controls. Both the intestinal absorption and fluid transfer using the different solutes were similar to those previously described (Madge 1976a, b) and hence the results will not be described in detail. Disaccharide hydrolysis and fluid transfer. Figure 3 also gives the absorption of D-glucose following hydrolysis of o-maltose by membrane-bound maltase and tissue accumulation of D-glucose. The results of the PCDF-treated mice and those of the controls were virtually identical. The fluid transfer (Table I) was also close. Exogenous energy supply and fluid transfer. Signific a n t differences in D-glucose absorption between PCDF-treated animals and the controls were not found when D-mannose was included in the serosal

Hishdme

Orndhme

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Monnose (=glucose)

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Fig, 3. Intestinal absorption of difl'erent solutes 7 days after a single dose of chlorinated dibenzofuran at a concentration of 2.0 mg kg-1 body wt. Each set of histograms represents the final mucosal (M), serosal (S) and supernatant of gut tissue homogenate (H) concentrations. For each solute, the two sets of histograms represent on the left the treated animals and on the right the controls. Initial concentration of sugars 8.0m-molar and that of amino acids 2.0m-molar. Means of 8-10 sacs; for clarity the S.E.M. have been omitted. Further details in text.

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Fig. 4. Intestinal absorption of different solutes 7 days after a single dose of non-chlorinated dibenzofuran at a concentration of 2.0 mg kg- ~ body wt. Other details as in Fig. 3. fluid (Fig. 3). Howevcr, the final tissue accumulation of D-glucose was slightly' higher in the controls than in the treated mice (P = 0.02-0.051. The tissue fluid transfer remained unaltered (Table 1). (2)

Non-chlorinated dihenzofuran (NCDF)

Figure 4 compares the effects of NC'DF-treated animals with untreated controls 7 days after a single dose of 2.0 mg kg- ' body wt each. Using D-glucose, intestinal absorption was significantly reduced (P = 0.01 0.0011 with treated mice: tissue accumulation of D-glucose also decreased slightly (P = 0.05). With the remaining solutes significant differences in intestinal absorption between treated and nontreated animals were not found. The absorption of D-glucose following membrane hydrolysis of tymaltose or after adding t)-mannose to the serosal fluid also remained unchanged. However, the tissue ac-

cumulation of [~glucose when exogenous energy (D-mannose) was provided was slightly lowered in the treated animals (P = 0.02.-0.05). Significant differences in serosal fluid transfer or gut fluid uptake between NCDF-treated animals and the controls were not found (results not given).

Polychlorinated naphthalenes (PCNsl Effect of different dosages. Figure 5 shows

(3)

the intestinal absorption of D-glucose 7 days after treatment with PCN (50!';, chlorine content) at different concentrations. Using a dose of 0.50 g kg- ' body wt, absorption remained unaltered: using a dose of 0.75 g kgit decreased slightly (P = 0.02-0.05L and following doses of 1.0, 2.0 and 3.0 g kg-~ body wt absorption was significantly decreased (P = 0.01 I. The final tissue accumulation of D-glucose at doses of 1.0, 2.0 and 3 . 0 g k g -a body wt averaged 0.49_+0.10m-molar:

Table I. Fluid transfer in mouse c',erted whole small intestine 7 days after a single d o ~ (2.0mg k g ~ bod.,, wt) of chlorinated dibenzofuran using different solutes. (Results using D-glucose also included.) Means of 8 10 sacs ± S.E.M. Results expressed as absolute values. Solute

Initial gut wet weight (gl

Serosal fluid transfer (g)

Gut fluid uptake tgl

~" PCDF [ Control

1.51 ± 0.11 1.56 + 0.10

1.97 _+ 0.15 1.99 _+ 0.23

0.76 ± 0.10 0.68 _+ 0.09

PCDF Control

1.53 + 0.09 1.48 +_ 0.16

0.68 _+ 0.07 0.61 +_ 0.09

0.49 _+ 0.06 0.34 _+ 0.05

L-Arginine

~" PCDF [ Control

1.51 _+ 0.13 1.46 _+ 0.14

0.48 _+ 0.07 0.42 _+ 0.06

0.29 _+ 0.07 0.24 _+ 0.06

e-Histidine

fPCDF [Control

1.52 _+ 0.09 1.44 +_ 0.07

0.52 _+ 0.09 0.53 _+ 0.06

0.34 _+ 0.08 0.30 _+ 0.05

l.-Ornithine

~'PCI)[-" [Control

1.51 _+ 0.10 1.66 4- 0.11

0.55 _+ 0.06 0.47 + 0.07

0.23 _+ 0.05 0.29 + 0.06

L-Proline

/PCDF [Control

1.58 + 0.13 1.67 _+ 0.09

0.33 + 0.06 0.31 _+ 0.07

0.32 _+ 0.07 0.36 + 0.08

~'PCDF [Control ~'PCDF [Control

1.45 1.49 1.57 1.46

2.17 2.13 2.44 2.36

0.68 0.64 0.82 0.78

D-Glucose t)-Galactose

I)-Mahose (D-Glucose) I)-Mannose

Treatment

_+ 0.13 _+ 0.12 + 0.14 _+ 0.11

_+ 0.20 _4- 0.31 _+ 0.12 +_ 0.14

+ 0.09 +_ 0.06 + 0.10 +_ 0.08

Action of dibenzofurans and naphthalenes 20 co

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Fig. 5. Intestinal absorption of D-glucose 7 days after a single dose of polychlorinated naphthalene (chlorine content 50%) at different concentrations. Other details as in Fig. I.

26

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that of the controls was 1.8 + 0.08m-molar. The serosal fluid transfer and gut fluid uptake remained unchanged (results not given). Effect of chlorine contents. The effects of PCNs having a range of 7 different chlorine contents (dose 1.0g k g - , body wt) on intestinal absorption of Dglucose are summarized in Fig. 6. Absorption of Dglucose was uniformly decreased (P = 0.01) in all the experiments, suggesting that there was no relationship between chlorine content and malabsorption. The serosal fluid transfer and gut fluid uptake remained unaltered (results not given). Absorption of other .solutes and fluid transfer. Figure 7 summarizes the effects of PCNs at different chlorine contents on intestinal absorption of o-galactose, L-arginine and L-histidine; also included are the final tissue accumulation of these solutes. Generally, the differences between the PCN-treated animals and those of the controls were small and not significant. The serosal fluid transfer and gut fluid uptake were also not significant (results not given).

Disaccharide

r"

10

62 70 C Fig. 6. Intestinal absorption of D-glucose 7 days after a single dose (l.0g kg-t body wt) of polychlorinated naphthalenes at different chlorine contents (%). Other details as in Fig. I. 56

The fluid transfer remained unaltered (results not given). Exogenous energy supply and fluid transfer. The increased D-glucose absorption in the presence of D-mannose in the serosal fluid in PCN-treated mice remained unchanged compared with the controls (Fig. 7). The tissue accumulation of D-glucose, however, was slightly lower in the treated animals than in the controls (P = 0.02-0.05). The fluid transfer in the experiments was unchanged (results not given).

hydrolysis and fluid transfer. The

absorption of D-glucose following hydrolysis of D-mahose by membrane-bound maltase in both PCN-treated animals and the controls was similar. as was the tissue accumulation of o-glucose (Fig. 7).

E

This work investigated in vitro small intestinal absorption of several monosaccharides and amino Maltose (=glucose)

Galactose

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Fig. 7. Intestinal absorption of different solutes 7 days after a single dose of polychlorinated naphthalenes at different chlorine contents (%L each at a concentration of 1.0g k g - ' body wt. Other details as in Fig. 3.

366

DAVID S. MADGI:

acids and concomitant fluid transfer in mice following a single, oral dose of PCDF, NCDF, and PCNs having 7 different chlorine contents. Each chemical signiticantly reduced the absorption of D-glucose while the absorption of f~-galactose and the amino acids remained unaltered compared with untreated controls. D-Glucose is both actively absorbed and metabolized, while l~-galactose and the amino acids are actively absorbed but not metabolized. Since oglucose and o-galactose both share a common carrier at the brush border membrane the results suggest that the carrier function of the brush border membrane was unimpaired in the treated animals. Using the disaccharide t~-maltose as solute the hydrolytic product. t~-glucosc, absorbed by both treated mice and the controls was unchanged, suggesting that membrane digestion at the brush border was unaffected. When an exogenous supply of energy, o-mannosc, was added to the serosal fluid, the final amount of Dglucose absorbed by the treated and control mice was similar, suggesting that the malabsorption of Dglucose without an energy supply was the result of l~-glucose being metabolized during the experiment. The final accumulation of o-glucose in the intestinal tissues was decreased in the treated animals but that of the other solutes remained similar to that of the controls, also suggesting that D-glucose was being metabolized during the experiment. The amount of D-glucose left in the mucosal fluid at the end of the experiments in both the treated and control animals suggested that the same amount o( D-glucose was being initially transported. In all the experiments, the fluid transfer was unchanged, and any alterations to the histology of the small intestine were minimal. Hence. it is concluded that the apparent lowered o-glucose absorption in the treated animals was probably the result of enhanced metabolism of o-glucose in the affected tissues and was not caused by malfunction of the absorptive cells at the brush border membrane. Similar effects on small intestinal function were observed in mice given commercial preparations of PCBs (Madge, 1976a, b) and P('Ts (Madge, 1977a). For example, using the PCB Aroclor 1260 and Phenoelor DP6 at single doses of 1.0 g kg- ~ body wt, D-glucose absorption fell by 27"~, and 45% respectively after 7 days; using the PCT Aroclor 5460 at single doses of 1.0 g kg-~ body wt, o-glucose absorption decreased by 36°i, after 7 days. The absorption of other solutes was unaffected. In the present work, the isolated toxic impurities in PCBs and PCTs also resulted in significant o-glucose malabsorption. Thus, with PCDF and NCDF the absorption of D-glucose was decreased by 45°0 and 370, respectively, with PCNs it fell by an average of 24'j,;. However, amounts of PCDF, NCDF and PCNs given in each dose which led to D-glucose malabsorption differed widely. Using PCDF or NCDF, a dose as low as 2.0 mg kg- J body wt resulted in pronounced D-glucose malabsorption, but with PCNs the dose had to be increased to 1.0gkg-~ body wt to give impaired absorption. Hence it is concluded that the malabsorptive effects caused by PCBs, and possibly PCTs, were largely the result of PCDF and, if present, NCDF impurities, while PCN impurities were virtually ineffective at comparable doses.

The toxicity of PCDF and NCDF and PCNs in commercial preparations of PCBs have also been studied by other workers. Vos et al. (1970) found significant differences in toxicity between three commercial PCB compounds containing 60°,, chlorine: Clophen 60, Phenoclor DP6 and Aroclor 1260. For example, when Clophen, Phenoclor and Aroclor were injected into the air space of chicken eggs. 0. 5 and 80°o of the eggs hatched respectively. Using microchemical analyses, traces of chlorinated penta- and dibenzofurans were identified in the first two compounds (5 ppm and 20ppm respectively in 2.0 mg samples) but neither was found in the third. Other experiments (Vos & Becms, 1971) confirmed that skin lesions and liver damaged in rabbits were caused mainly by chlorinated dibenzofuran contaminants in commercial PCB preparations and to a minor extent by the PCBs themselves. Hoffman (1958) found that a single dose (0.5 1.0mgkg -~ body wt) of tetrapentachlorodibenzofuran in rabbits caused severe liver damage, whereas chlorinated naphthalenes were far less toxic. Traces of chlorinated dibenzofurans, as distinct from PCBs or PCTs. have been isolated in wildlife populations of herring gull eggs and in the liver of sea-lions (Bowes et al.. 1973). and from muscle and liver of immature brook trout after being given doses of these chemicals (Zitko et al.. 1973). Other work (Madge. 1977b) has also shown that traces of impurities in commercial preparations of a widely used compound were mainly responsible for malabsorptive effects. Following single doses of 25-250 mg kg- ~ body wt of purified 2.4.5-T (trichlorophenoxyacetic acid), a commercial herbicide, absorption of D-glucose decreased progressively the higher the dosage. Experiments were also made on purified chlorodioxins, which are impurities formed during the commercial preparation of the herbicide. TCDD (2.3.7,8-tetrachlorodibenzo-p-dioxin). the main impurity in 2.4,5-'I. markedly reduced D-glucose absorption at considerably lower single doses of 25- 300 ~ug kg -z body wt. DCDD (2.7-dichlorodibenzo-p-dioxin) and OCDD (I.2.3.4.6.7,8.9-octachlorodibenzo-p-dioxin) gave similar effects using D-glucose. but at relatively higher doses of 2.0--10.0mgkg' body wt. These experiments, and the results of others, on the effects of these toxic contaminants found in the herbicide were analagous to the effects of the isolated contaminants in PCB and possibly PCT preparations used in the present study, that is, the apparent malabsorption of o-glucose was probably the result of enhanced I~-glucose metabolism while the digestive and absorptive functions of the brush-border membrane remained unimpaired.

Acknowledgements--I am grateful to Miss Jacqueline Benson for her technical assistance. Dr A. M. Scofield for criticizing the manuscript, and Miss Dani Macintyre for typing it.

RI'~FERENCES

BAILE', N. T. J. (1959) Statistical Methods in Biolo.qy. English Universities Press. London. Brat L. M. & HtRD F. J. R. (19561 The uptake of amino acids by carrot slices. Biochem. J. 64, 305--311.

Action of dibenzofurans and naphthalenes BOWLSG. W., SIMONEITB. R., BURLINGAMEA. L.. DE LAPPE B. W. & RISEBROUGHR. W. (1973) The search for chlorinated dibenzofurans and chlorinated dibenzoidioxins in wildlife populations showing elevated levels of embryonic death. Em:ir. HIth Perspect. exp. Issue 5, 191-198. CmNARD F. P. (1952) Photometric estimation of proline and ornithine. J. biol. Chem. 199, 91-95. FISHBEIN L. (1974) Toxicity of chlorinated biphenyls. A. Retd. Pharmac. 14, 139-156. HOFFMANN H. T. (1958) Neuere Erfahrungen mit hochtoxischen Chlorkohlenwasserstoffen. Nauryn-Schmeideberys" • Arch. exp. Path. Pharmak. 232, 228-230. MACPHERSON T. H. (1946) The basic amino-acid content of proteins. Biochem. J. 40, 470-481. MAOGE D. S. (1976a) Polychlorinated biphenyls and intestinal absorption of D-gluco~ in mice. Gen. Pharmac. 7, 45-48. MADGE D. S. (1976b) Polychlorinated biphenyls (Phenoclot and Pyralene) and intestinal transport of hexoses and amino acids in mice. Gen. Pharmac. 7, 249-254. MAtX~E D. S. (1977a) Polychlorinated terphenyls and intestinal transport in mice. Gen. Pharmac. 8. 43-46.

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MADGE D. S. (1977b) Effects of trichlorophenoxyacetic acid and chlorodioxins on small intestinal function. Gen. Pharmac. 8, 319-324. MORLEY G., DAWSON A. & MARKS V. (1968) Manual and autoanalyzer methods for measuring blood glucose using guaiacum and glucose oxidase. Proc. Ass. clin. Biochem. 5, 42-45. SOMOGYI M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-23. Vos J. G. (1972) Toxicology of PCBs for mammals and for birds. Envir. Hlth Perspect. exp. Issue !, 105-117. Vos J. G. & BEEMS R. B. (1971) Dermal toxicity studies of technical polychlorinated biphenyls and fractions thereof in rabbits. Toxic. appl. Pharmac. 19, 617-633. VOS J. G.. KOEMANJ. H., VAN DER MAASH. L., DE BRAUW M. C. & DE VOS R. H. (1970) Identification and toxicological evaluation of chlorinated debenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenyls. Fd. Cosmet. Toxic. 8, 625-633. ZITKO V., WILDISH D. J., HUTZINGER O. 8~ CHOI P. M. K. (1973) Acute and chronic oral toxicity of chlorinated dibenzofurans to ~lmonid fishes. EnFir. HIth Perspect. exp. Issue. 5, 187-189.