~oxrc m Vim Vol 3, No I, pp 77-81, 1989 0887-2333/89 $3.00 + 0.00 Pnnted m Great Bntam Pergamon Press plc EFFECT OF MONO-(2-ETHYLHEXYL) PHTHALATE ON THE METABOLISM OF ENERGY-YIELDING SUBSTRATES IN RAT SERTOLI CELL-ENRICHED CULTURES N. R. WORRELL, W. M. COOK, C. A. THOMP~~N and T. J. B. GRAY British Industnal Blologlcal Research Assoclatlon, Woodmansterne Road, Carshalton, Surrey SM5 4DS, England (Recezved 17 March 1988; rezmons recezved 5 July 1988) Abstract-The effect of the testicular toxm mono-(2-ethylhexyl) phthalate (MEHP) on the metabohsm of energy-yielding substrates m Sertob cell-ennched cultures has been studled MEHP stimulated glucose uhhzatlon and oxldatlon. Stimulation of “C0, production was greater with [1-‘4C]- than with [6-14C]glucose Oxidation of [l%]pyruvate and [U-‘4C]acetate to 14C02 was reduced by MEHP treatment m the presence but unaffected m the absence of glucose MEHP increased the mcorporation of radioactivlty from [1-14C]- and [6-14C]glucose but not from [U-‘4C]acetate into fatty aads. MEHP markedly mcreased the production of lactate by Sertoh cells cultured m the presence of 5.5 mh+glucose and 0 5 mM-pyruvate Decreasmg the glucose concentration reduced this stlmulatory effect In glucose-free medium contammg 0 5 or 2.5 mM-pyruvate MEHP had no effect on Sertoli cell lactate production. Thus, MEHP appears to stimulate the uhhzatlon of glucose by the Sertoh cell but does not appear to have a direct effect on the converSion of pyruvate to lactate, on the converslon of pyruvate to acetyl CoA or a direct effect on the metabohsm of acetyl CoA through the Krebs cycle Increased glucose utlhzatlon appears to be a consequence of the increased metabolism of glucose that occurs through the glycolytlc and pentose phosphate pathways. These results suggest that m contrast to Its effects on the hver, MEHP does not exert a pnmary effect on the Sertoh cell mltochondnon INTRODUCTION Phthalate diesters are widely used in various chemical industries as plasticizers and solvents. Certain phthalate diesters produce testicular damage when administered to rodents (Foster et al. 1980), and although the lesion is characterized by shedding of spermatocytes and spermatids from the germinal epithelium, more recent mvestigations of function and morphology have mdicated that the Sertoh cell may be the initial site of injury (Creasy et al. 1983; Gray & Gangolli, 1986). Orally administered phthalate diesters are metabo- lized in the gut to the corresponding monoesters (Albro, 1987). The available evidence suggests that these monoesters are responsible for the toxic effects in the testis (Sjiiberg et al 1986). When co-cultures of Sertoh and germ cells are exposed to these toxic monoesters, detachment of the germ cells from the underlymg Sertoli cell monolayer is observed, which suggests that the lesion that occurs in vivo can be modelled in vitro (Gray & Beamand, 1984). In addi- tion, Creasy et nl. (1988) have demonstrated that there are marked similarities in the ultrastructural response of Sertoli cells to phthalates in vivo and in vitro. Sertoli cells produce lactate and pyruvate as energy substrates for the developing germ cells (Grootegoed et al. 1984; Jutte et al. 1981). Recent studies (Moss et al. 1985 & 1988) have shown that the monoesters of Abbreviations: DMSO = dimethylsulphoxide; FSH = fol- hcle-stimulatmg hormone; MEHP = mono-(2-ethyl- hexyl) phthalate; PBS = phosphate-buffered saline those phthalate diesters that are potent testicular toxins in VIVO cause a significant increase in lactate secretion by Sertoli cell-enriched cultures. Pyruvate secretion is little affected so that the lactate to pyruvate ratio in the culture medium 1s markedly increased. This change in the extracellular lac- tate:pyruvate ratlo could have important imph- cations for the intracellular redox status of the cell. In order to investigate further the stlmulatory effect of phthalates on lactate production by rat Sertoh cells, studies on the effects of mono-(Z ethylhexyl) phthalate (MEHP; one of the most potent phthalate monoesters in eliciting germ-cell detach- ment in vztro) on the metabolism of energy yielding substrates were undertaken. Since Sertoh cells com- prise less than 3% of the cells in the mature testis the use of Sertoli cell-enriched cultures facilitated this investigation. MATERIALS AND METHODS Materials. D-[5-3H]glucose (sp. act. 15.7 Ci/mmol), D-[l-14C]glucose (sp. act. 59 mCi/mmol), D-[6-14C]- glucose (sp. act. 58.5 mCi/mmol), [U-14C]acetate (sp. act. 58 mCi/mmol) and [l-14C]pyruvate (sp. act. 28 mCi/mmol) were purchased from Amersham International plc, Amersham, Bucks. MEHP (purity > 99% by high-pressure liquid chromatography) was synthesized by the method of Albro et al. (1973), and the identity was confirmed by nuclear magnetic reso- nance and elemental analysis. Cell culture materials were purchased from Gibco, Paisley, Scotland. Preparation of Sertoli cell-enriched cultures. Mixed cultures of Sertoli and germ cells were prepared from 77
Transcript
~oxrc m Vim Vol 3, No I, pp 77-81, 1989 0887-2333/89 $3.00 + 0.00 Pnnted m Great Bntam Pergamon Press plc
EFFECT OF MONO-(2-ETHYLHEXYL) PHTHALATE ON THE METABOLISM OF ENERGY-YIELDING SUBSTRATES
IN RAT SERTOLI CELL-ENRICHED CULTURES
N. R. WORRELL, W. M. COOK, C. A. THOMP~~N and T. J. B. GRAY British Industnal Blologlcal Research Assoclatlon, Woodmansterne Road, Carshalton, Surrey SM5 4DS,
England
(Recezved 17 March 1988; rezmons recezved 5 July 1988)
Abstract-The effect of the testicular toxm mono-(2-ethylhexyl) phthalate (MEHP) on the metabohsm of energy-yielding substrates m Sertob cell-ennched cultures has been studled MEHP stimulated glucose uhhzatlon and oxldatlon. Stimulation of “C0, production was greater with [1-‘4C]- than with [6-14C]glucose Oxidation of [l%]pyruvate and [U-‘4C]acetate to 14C02 was reduced by MEHP treatment m the presence but unaffected m the absence of glucose MEHP increased the mcorporation of radioactivlty from [1-14C]- and [6-14C]glucose but not from [U-‘4C]acetate into fatty aads. MEHP markedly mcreased the production of lactate by Sertoh cells cultured m the presence of 5.5 mh+glucose and 0 5 mM-pyruvate Decreasmg the glucose concentration reduced this stlmulatory effect In glucose-free medium contammg 0 5 or 2.5 mM-pyruvate MEHP had no effect on Sertoli cell lactate production. Thus, MEHP appears to stimulate the uhhzatlon of glucose by the Sertoh cell but does not appear to have a direct effect on the converSion of pyruvate to lactate, on the converslon of pyruvate to acetyl CoA or a direct effect on the metabohsm of acetyl CoA through the Krebs cycle Increased glucose utlhzatlon appears to be a consequence of the increased metabolism of glucose that occurs through the glycolytlc and pentose phosphate pathways. These results suggest that m contrast to Its effects on the hver, MEHP does not exert a pnmary effect on the Sertoh cell mltochondnon
INTRODUCTION
Phthalate diesters are widely used in various chemical industries as plasticizers and solvents. Certain phthalate diesters produce testicular damage when administered to rodents (Foster et al. 1980), and although the lesion is characterized by shedding of spermatocytes and spermatids from the germinal epithelium, more recent mvestigations of function and morphology have mdicated that the Sertoh cell may be the initial site of injury (Creasy et al. 1983; Gray & Gangolli, 1986).
Orally administered phthalate diesters are metabo- lized in the gut to the corresponding monoesters (Albro, 1987). The available evidence suggests that these monoesters are responsible for the toxic effects in the testis (Sjiiberg et al 1986). When co-cultures of Sertoh and germ cells are exposed to these toxic monoesters, detachment of the germ cells from the underlymg Sertoli cell monolayer is observed, which suggests that the lesion that occurs in vivo can be modelled in vitro (Gray & Beamand, 1984). In addi- tion, Creasy et nl. (1988) have demonstrated that there are marked similarities in the ultrastructural response of Sertoli cells to phthalates in vivo and in vitro.
Sertoli cells produce lactate and pyruvate as energy substrates for the developing germ cells (Grootegoed et al. 1984; Jutte et al. 1981). Recent studies (Moss et al. 1985 & 1988) have shown that the monoesters of
those phthalate diesters that are potent testicular toxins in VIVO cause a significant increase in lactate secretion by Sertoli cell-enriched cultures. Pyruvate secretion is little affected so that the lactate to pyruvate ratio in the culture medium 1s markedly increased. This change in the extracellular lac- tate:pyruvate ratlo could have important imph- cations for the intracellular redox status of the cell.
In order to investigate further the stlmulatory effect of phthalates on lactate production by rat Sertoh cells, studies on the effects of mono-(Z ethylhexyl) phthalate (MEHP; one of the most potent phthalate monoesters in eliciting germ-cell detach- ment in vztro) on the metabolism of energy yielding substrates were undertaken. Since Sertoh cells com- prise less than 3% of the cells in the mature testis the use of Sertoli cell-enriched cultures facilitated this investigation.
MATERIALS AND METHODS
Materials. D-[5-3H]glucose (sp. act. 15.7 Ci/mmol), D-[l-14C]glucose (sp. act. 59 mCi/mmol), D-[6-14C]- glucose (sp. act. 58.5 mCi/mmol), [U-14C]acetate (sp. act. 58 mCi/mmol) and [l-14C]pyruvate (sp. act. 28 mCi/mmol) were purchased from Amersham International plc, Amersham, Bucks. MEHP (purity > 99% by high-pressure liquid chromatography) was synthesized by the method of Albro et al. (1973), and the identity was confirmed by nuclear magnetic reso- nance and elemental analysis. Cell culture materials were purchased from Gibco, Paisley, Scotland.
Preparation of Sertoli cell-enriched cultures. Mixed cultures of Sertoli and germ cells were prepared from
77
78 N R WORRELL et al
Table I Effect of mono-(2-ethylhexyl) phthalate (MEHP) on the metabohsm of glucose by Sertoh cell-ennched cultures
Substrate converted to ‘H,O or ‘“CO, (nmol/mg protem)
Substrate Control MEHP % Control
5-[‘HJglucose 448k31 749 k 49”. 167 I-[l~C]glucose 313+52 75 6 k 3 3**’ 242 6-[‘4C]glucose 25Oi13 30 3 f 1 2’ 121
5-[‘HIglucose was present at a radlochemlcal concentration of 1 bCl/ml, whde I-[“Cl- and 6-[‘4C]glucose were present at a radlochemtcal concentratton of 0 25 pCl/ml All cultures contamed a final glucose concentration of 5 5 rnM Cultures were mcubated for 3 hr wth or wtthout MEHP (100~~) Values are means f SEM for 4 culture dishes from smgle experiments typlcal of 24 rephcates wth different cell preparations (*P < 0 05, ***P < 0 001, least slgmficant dG.xence test)
28-day-old Sprague-Dawley rats as previously de- scribed (Gray & Beamand, 1984) and maintained at 32°C m 25 cm* tissue culture flasks. Cells were main- tamed for 24 hr m Eagle’s mmlmal essential medium supplemented with 4 mM+glutamine, 0 1 mM-non- essential amino acids, 100 U pemctlhn/ml, 100 pg streptomycm/ml and 10% foetal calf serum. There- after, cells were mamtamed m serum-free medium, which was changed every 24 hr. After 72 hr, germ cells were removed by treatment w’th hypotomc Tns buffer (20m~-Tns-HCl, pH 7 4) for 5 min as described by Galdieri et al (1981). The resulting Sertoh cell-enriched cultures were then mamtamed m serum-free medium for a further 24 hr prior to metabohsm studies
Estwnation of substrate metabohsm The Sertoh cell-enriched cultures were washed twice with Dulbecco’s calcmm- and magnesium-free phosphate- buffered saline (PBS) and then incubated m sealed flasks for 3 hr m 4 ml PBS containing calcium chlo- nde (0.9 mM), magnesium chlortde (0.5 mM) and, unless otherwise stated, glucose (5.5 mM) with the appropriate radtolabelled substrate. MEHP (100 PM) was added as a solution m d’methylsulphoxide (DMSO), w’th control cultures receivmg an eqtuv- alent concentration (0.4%, v/v) of DMSO alone In the case of the [‘“Cl substrates, the mcubation was terminated by the mlection of 5 M-sulphurtc acid (0.5 ml) through the seal mto the medium. Ethanol- amme (0 5 ml) was then Injected through the seal into a small well suspended in the neck of the flask, and the flasks were incubated for a further 1 hr at room temperature to trap any [‘4C]carbon dioxide. The ethanolamme was them removed to a scmttllation vial, the well was washed twice with 2-ethoxyethanol (0.5 ml) and the washing added to the ethanolamine, and scmtillant (10 ml; Optiphase Safe, LKB Scintil- lation Products, Fisons, Loughborough, Le’cs.) was added. In the case of the [5-3H]glucose, the incu- batton was terminated by the addition of 1 M-HCl (0.4ml) to the medium. Medium (0.5 ml) was then transferred to a plastic well suspended m a 20 ml counting vial contammg 0.3 g anhydrous calcium chloride. The vial was sealed and incubated at 45-50°C overnight. The well was removed and water (1 ml) and scintillant (15 ml; Instagel, Canberra Packard, Pangbourne, Berks ) were added to the vial.
The Sertoli cell monolayer was washed twice with PBS. The cells were removed from the dishes using a rubber pohceman and then somcated m water. Samples were taken for protein esttmation and fatty
acid extractton. Fatty acids were extracted according to the method of Dole & Meinertz (1960).
Biochemical estimations. Lactate was measured with the method of Hohorst (1970). Protein esti- mations were made using the method of Lowry et al. (1951)
RESULTS
All observattons were made using a treatment level of 100~~-MEHP. This concentration was selected smce it had been shown to produce clear effects in vitro but was not high enough to produce non-specific mamfestattons of cytotoxicity (Creasy et al. 1988; Gray & Beamand, 1984, Moss et al. 1988).
The effect of MEHP on the metabohsm of 5j3H]-, 1-[‘4C]- and 6-[‘4C]glucose m Sertoli cell-enriched cultures 1s shown in Table 1 The metabolism of 5-[3H]glucose to [‘HIwater, and the metabohsm of both 1-[‘4C]- and 6-[‘4C]glucose to [‘4C]carbon dioxtde were all stattstically sigmficantly increased by treatment with 100~~-MEHP for 3 hr to 167, 242 and 121% of the control values, respectively.
Table 2 summarizes the effect of 100~~-MEHP treatment on the oxidation of l-[i4C]pyruvate and U-[‘4C]acetate. In the presence of 5.5 mM-glucose the conversion of both pyruvate and acetate to [‘4C]carbon dioxide was significantly decreased by treatment for 3 hr with 100 PM-MEHP. When glucose was omitted from the medium, MEHP had no effect on the oxidatton of either pyruvate or acetate.
Table 3 shows the effect of 100 PM-MEHP on the mcorporation of radioactivity from 1-[“Cl- and 6-[‘4C]glucose and U-[‘4C]acetate into Sertoli cell fatty acid Incorporation of radtolabel mto fatty acids was significantly increased by 100 PM-MEHP treat- ment for 3 hr when either 1-[‘4C]- or 6-[‘4C]glucose was present m the cultures (263 and 202% of control values, respectively) but unaffected when cultures were incubated with U-[‘4C]acetate. The effect of 100~~-MEHP treatment for 3 hr on lactate prod- uction by Sertoli cell cultures incubated in media containing varying amounts of glucose and pyruvate is summarized in Table 4. MEHP elicited a marked sttmulatton of lactate production in cultures mcu- bated in PBS supplemented with 5.5 mw-glucose (288% of control). Addition of 0.5mmpyruvate to the medium slightly decreased the extent of th’s stimulation. Decreasing the glucose concentration to 0.55 mM while maintaming the pyruvate concen- tration at 0 5 mM further reduced the stimulatton of
Phthalate monoesters and Sertoli cell metabohsm 19
Table 2 Effect of mono-(2-ethylhexyl) phthalate (MEHP) on the metabohsm of pyruvate and acetate by Sertob cell-ennched cultures
Substrate converted to “CO, (nmol/mg protem)
Wl Glucose Substrate concn (mhi) Control MEHP (100~~) % Control
1-[“Clpyruvate was present at a final concentration of 10~~ and 025pCl/ml, U-[“Clacetate was present at a final concentration of 5 PM and 0 25 pCl/ml Cultures were Incubated for 3 hr with or wIthout MEHP (100 PM). Values are means + SEM for 4 culture dishes from smgle expenments typIca of 24 rephcates with different cell preparations (‘P < 0 05, least srgmficant difference test)
lactate production by MEHP, and at a glucose concentration of 0.055 mM, MEHP had no effect on lactate production. There was also no stimulation of lactate secretion by MEHP when cultures were incubated with either 0.5 or 2.5 mM-pyruvate in the absence of glucose
DISCUSSION
Labelled glucose, pyruvate and acetate have been used to evaluate the contnbution of different path- ways to the metabolism of glucose in Sertoli cells and to determine how this is affected by concentrations of MEHP known to stimulate lactate secretion and to elicit characteristic morphological changes in these cells (Creasy et al. 1988; Moss et al. 1988).
The formation of [3H]water from [5-3H]glucose 1s a measure of the overall utilization of glucose both by glycolysis and by the pentose phosphate pathway. The formation of 14C0, from [6-14C]glucose measures flux through glycolysis and subsequent oxidation by Krebs cycle enzymes, while [I-14C]glucose also gives nse to 14C02 by this means and by metabohsm in the pentose phosphate pathway (Ashcroft et al 1972). In control cells, glucose was metabolized both by Krebs cycle oxidation and through the pentose phosphate pathway. Substantially more glucose was metabo- lized to glyceraldehyde-3-phosphate than underwent oxldatlon, which presumably reflects the metabolic function of Sertoli cells to provide lactate for germ-cell energy production (Jutte et al. 1981). These results are in good agreement mth those of
Table 3 Effect of mono-(2-ethylhexyl) phthalate (MEHP) on the mcorporatlon of radloactvzlty from (‘4C]-labelled substrates mto hpld m Sertoh cell-ennched
cultures
[14C] substrate converted to hpld (nmoljmg protem)
l-[‘4c]- and 6-[‘4c]glucose were added to grve a final radlochernzal concentration of 0 25 pQ/ml, U-[“Clacetate was added at a concentration of 5 PM and 0 25pC1/ml, unlabelled glucose was always present at a concentration of 5.5 mhi Cultures were Incubated for 3 hr with or without MEHP (100 PM) Values are means + SEM for 4 culture chshes from smgle expenments typIca of 2-4 rephcates wnh chfferent cell preparations (***F’ < 0001, least slgmficant chfference test)
Table 4. Effect of me&urn glucose and pyruvate concentrations on the snmulatory effect of mono-(2-ethylhexyl) phthalate (MEHP) on lactate productIon by Sertoh cell-ennched cultures
Lactate productIon Ad&Ion to me&urn @mo1/3 hr/mg protem)
Glucose Pyruvate MEHP (mt.0 IrnM) Control (100 UM) % Control
Cultures were Incubated for 3 hr cvlth or wtvlthout MEHP (100~~) Values are means k SEM for 4 culture dishes from smgle expenments typIcal of 2-l rephcates with different cell preparahons (*P < 0 05, **P < 0 01, ***P < 0 001, least slgmficant chfference test)
80 N. R WORRELL et al.
Grootegoed et al (1986), who found similar levels of glucose utilization and oxidation m Sertoli cells derived from rats irradiated with X-rays. Treatment of Sertoli cell cultures with MEHP resulted in a stimulation of glucose utilization and oxidation. The oxidation of [I-‘4C]glucose to r4C02 was increased to a greater extent than that of [6-14C]glucose, whtch suggests that MEHP also stimulates the pentose phosphate pathway in cultured Sertoh cells. These effects of MEHP on glucose utilization and oxidation by Sertoli cells m culture are similar to those of folhcle-stimulating hormone (FSH) (Grootegoed et al 1986). The reason for these similarities remains obscure but one explanation is that MEHP may act by affecting the FSH receptor pathway m the Sertoli cell.
The 14C radiolabel on [l-‘4C]pyruvate is lost to CO, m the step between pyruvate and acetyl CoA cata- lysed by pyruvate dehydrogenase while the radiolabel on [U-r4C]acetate is oxidized to CO2 during the Krebs cycle. In contrast to the evident stimulation of glu- cose metabolism, MEHP effected a decrease in both pyruvate and acetate metabolism when glucose was present in the incubatton medium, but had no effect on the metabolism of either substrate when glucose was absent (Table 2). The effect of MEHP when glucose was present m the medium could be due to alterattons in the intracellular pyruvate and acetate pools. If the formatton of unlabelled pyruvate and acetate were increased, dilution of the radiolabelled material and an apparent reduction m its metabolism would result. Since MEHP stimulates the utilization of glucose tt 1s possible that the mtracellular pyruvate and acetate pools are mdeed altered. The lack of effect of MEHP on pyruvate and acetate metabolism m the absence of glucose supports this. These results suggest that the conversion of pyruvate to acetyl CoA and the subsequent Krebs cycle reactions are unaffected by MEHP. Chapin et al (1988), m agree- ment with our findings, also found a decrease m the conversion of [U-r4C]acetate to r4C02. Their studies were conducted m the presence of glucose only.
MEHP stimulated the mcorporation of radiolabel from both [1-r4C]- and [6-i4C]glucose into fatty acids, whereas it had no effect on the incorporation of radtolabel from [U-r4C]acetate These dtfferences may again be a result of alterations in intracellular pre- cursor pools. In the case of the incubations with [r4C]glucose, increased glycolysis would increase the [14C]acetate pool size in the treated cells. This could lead to an overestimatton of the rate of fatty acid biosynthesis. Conversely, in the case of the mcu- battons with [r4C]acetate, increased glycolysis would increase the concentration of cold acetate present in the precursor pools leading to an apparent decrease in fatty acid biosynthesis. Chapin et al. (1988) found an increase in neutral lipid m cells treated with MEHP, which suggests that increased fatty acid biosynthesis may well occur
Decreasing the glucose concentration in the mcu- bation medium reduced the stimulation of lactate secretion by MEHP without appreciably affecting the basal lactate output by the control cultures (Table 4). This suggests that MEHP does not directly affect the reduction of pyruvate to lactate by lactate dehydrogenase.
From Tables 1 and 4 it can be calculated that m untreated cultures, allowmg for the amount of glucose undergoing complete oxidation, the lactate secreted into the culture medium represents approxi- mately 67% of the utilized glucose. If a similar calculation is performed for MEHP treated cultures the amount of lactate secreted in the culture medium exceeds the amount of glucose utilized by approxi- mately 20%. The source of the additional lactate 1s not known.
Thus, the stimulation of lactate secretion by MEHP does not appear to result from a direct actton on the reduction of pyruvate to lactate nor from a block m the subsequent metabolism of pyruvate by Krebs cycle enzymes. It appears that MEHP exerts a stimulatory effect at some pomt or points in gly- colysis and/or the pentose phosphate pathway prior to pyruvate formation. Chapm et al (1988), in- vestigating the effect of MEHP treatment on Sertoli cell succinate dehydrogenase, found no effect when the enzyme assay was performed m the absence of MEHP and a slight inhibition when it was present. They concluded that Sertoh cell mitochondria may be a target for MEHP toxictty. Studies of the hepatic effects of MEHP have also suggested that the mito- chondna are the putative targets for toxicity in this organ (Melnick & Schiller, 1985) Our results suggest that mitochondrta are unlikely to be the primary target organelle m the Sertoli cell. In addttion, Chapm et af (1988) found that MEHP had no effect on Sertoh cell ATP levels durmg the first 8 hr after exposure. This is consistent with our conclusion that the increased lactate production is not a consequence of impaired mitochondrial function.
A number of other testicular toxicants, mcludmg lead, dibromochloropropane and gossypol, have also been shown to alter lactate productton by cultured Sertoli cells (Batarseh et al. 1986; Miller et al. 1985, Reyes et al. 1986). Studies have shown that m the case of dibromochloropropane and gossypol the alter- ation in lactate secretion IS thought to be a consequence of the uncoupling of oxidative phos- phorylatton by these agents. Our studtes suggest that a different mechanism is responsible for the stimulation of lactate secretion elicited by MEHP
Acknowledgements-The authors wash to thank Mrs Angela Savage for the preparatton and mamtenance of the cell cultures, Dr R Purchase for synthesizing the MEHP and Dr S. D. Gangolh (Dtrector of Research) for support and encouragement Thts work forms part of a research proJect sponsored by the UK Mmtstry of Agrtculture, Ftshertes and Food to whom our thanks are due. The results of the research are the property of the Mmtstry of Agrtculture, Ftshertes and Food and are Crown copyright
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