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*
School of Food and Nutritional Sciences, The University of Shizuoka, Shizuoka 422-8526, Japan; and
Department of Applied Molecular Biosciences, Nagoya University, Nagoya 464-8601, Japan
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • taurine • polychlorinated biphenyls • ascorbic acid • cytochrome P-450 • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
ii) elevation of serum levels of HDL-cholesterol and apolipoprotein
A-I (Oda et al. 1990
, Oda and Yoshida 1994
), iii) accumulation of liver lipids (Kato et al. 1980a
, Oda et al. 1994
) and iv) enhancement of
ascorbic acid in urine and tissues (Horio and Yoshida 1982
, Kato et al. 1980b
).
Changes in ascorbic acid metabolism by xenobiotics have been known
since 1940s (Burns et al. 1954
and Burns et al. 1957
, Conney and Burns 1959
,
Hollmann and Touster 1962
, Horio and Yoshida 1982
, Kato et al. 1980
, Lake et al. 1978
, Longenecker et al. 1940
). Treatment of
rats with xenobiotics enhances the turnover rate of ascorbic acid and
ascorbic acid biosynthesis through the D-glucuronate
pathway (Burns et al. 1954
, Hollmann and Touster 1962
, Horio and Yoshida 1982
, Horio et al. 1983
). Alterations in ascorbic acid metabolism were
correlated with the induction of drug-metabolizing enzymes in rats
(Conney and Burns 1959
). Some enzymes in the
D-glucuronate pathway were induced by xenobiotics such as
UDP glucose dehydrogenase, UDP-glucuronosyltransferase (UDPGT), and
ß-glucuronidase (Hollmann and Touster 1962
,
Horio and Yoshida 1982
, Horio et al. 1983
). Because UDPGT, which catalyzes an important step of
ascorbic acid biosynthesis, is a phase II drug-metabolizing enzyme,
ascorbic acid metabolism is thought to be correlated with
drug-metabolism in xenobiotic-treated rats (Horio et al. 1993
). In guinea pigs, which are unable to synthesize ascorbic
acid like primates, treatment with ascorbic acid ameliorated the
toxicity of PCB (Kato et al. 1977
). Moreover, it has
been reported that ascorbic acid is required for drug metabolism
(Rikans et al. 1978
, Sato and Zannoni 1976
). Therefore, the enhancement of ascorbic acid synthesis
due to xenobiotics would be an adaptive and protective response to
exposure to xenobiotics.
Alterations of ascorbic acid and cholesterol metabolism by xenobiotics
are also influenced by dietary nutrients such as protein (Kato et al. 1980b
) and sulfur-containing amino acids (S-AA). For
example, in rats fed a diet containing a 100 g/kg soy protein isolate,
supplementation of L-methionine (13.4 or 33.5 mmol/kg diet)
and L-cystine (as half cystine, 13.4 or 33.5 mmol/kg diet)
significantly increased serum cholesterol level and urinary ascorbic
acid excretion (Oda et al. 1986
and Oda et al. 1989
). However, one of the end-products of
S-AA metabolism, sulfate, had no effect on serum cholesterol or
urinary ascorbic acid (Oda et al. 1986
). Whether another
metabolism end-product, taurine (2-amino ethanesulfonic acid),
influences metabolic changes such as ascorbic acid metabolism caused by
PCB has not been studied. Taurine affects various biological and
physiological functions, including cell membrane stabilization
(Pasantes-Morales et al. 1985
), antioxidation
(Nakamura et al. 1993
), detoxification, osmoregulation
(Huxtable 1992
), neuromodulation (Kuriyama 1980
) and brain and retinal development (Sturman 1986
). Our previous studies showed that feeding 5–50 g/kg
taurine for 2 wk enhanced the serum HDL-cholesterol concentration
in normal rats (Mochizuki et al. 1998
), and that feeding
taurine (0.25–50 g/kg diet) for 2 wk reduced serum level of
cholesterol in rats fed a high-cholesterol diet (Nanami et al. 1996
, Yokogoshi et al. 1999
).
We investigated the effect of taurine on the hepatic drug-metabolizing enzymes and the concentrations of ascorbic acid in tissues and urine in rats fed PCB to determine whether taurine has an adaptive and protective function in xenobiotic-treated rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Young male Wistar rats weighing about 60 g (Japan SLC, Hamamatsu,
Japan) were maintained at 24°C with a 12-h light (0700–1900) and
dark cycle. To accustom the rats to the experimental conditions, they
were initially allowed free access to a 200 g/kg diet of casein diet
(control diet) for 2 d and divided into four groups. Compositions
of test diets are shown in Table 1
. Rats were fed control diet, taurine-supplemented diet (30 g/kg
diet; Taisho Pharmaceutical Co., Ltd., Tokyo, Japan),
PCB-containing diet (0.2 g/kg diet; Arochlor 1254, Mitsubishi
Monsanto Co., Ltd., Tokyo, Japan) or PCB-containing diet
supplemented with taurine in a 2 x 2 factorial design. Taurine
and PCB were supplemented at the expense of carbohydrate. The rats were
individually housed in stainless cages in a room controlled for
temperature (23°C) and humidity (55%), and given free access to the
experimental diets and water for 14 d. The rats were killed by
decapitation at 1000 after 16 h of starvation on the last day in
the experiment, and blood was collected from the cervical wound.
Tissues were immediately removed, frozen on dry ice and then stored at
-80°C until assayed. The experimental procedures used in this study
met the guidelines of the Animal Care and Use Committee of the
University of Shizuoka.
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The serum lipids (total cholesterol and HDL-cholesterol) were
enzymatically measured by using commercial kits (cholesterol C-test
and HDL-cholesterol-test; Wako Pure Chemical, Osaka, Japan,
respectively). Urine was collected into 15 mL of 100 g/L metaphosphoric
acid solution to determine the concentrations of ascorbic acid.
Portions of liver and spleen were homogenized with 50 g/L
metaphosphoric acid solution and then centrifuged at 1500 x g for 10 min. The concentrations of ascorbic acid in the
metaphosphoric acid supernatant of tissue and urine were determined by
the 2,4-dinitrophenylhydrazine method (Roe and Kuether 1943
). Liver homogenate was prepared with ice-cold 150
mmol/L KCl in 10 mmol/L phosphate buffer, pH 7.4, with a
Potter-Elvehjem type Teflon homogenizer, and centrifuged at 10,000
x g for 10 min at 4°C. The postmitochondrial
supernatant was further centrifuged for 60 min at 105,000 x g at 4°C to obtain the microsomal pellets. Their pellets
were suspended in 150 mmol/L KCl, and the amounts of cytochrome P-450
and cytochrome b5 were determined by the method of Omura and Sato (1964)
.
Statistics.
Experimental data were statistically analyzed by two-way ANOVA. When the interaction (PCB x taurine) was significant (P < 0.05), Student’s t test was performed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Relative liver weight was unaltered by the supplementation of taurine
in rats that were not treated with PCB. However, in PCB-treated
group (PCB group), the relative liver weight was significantly greater
than that in C group (P < 0.001), and dietary taurine
amplified its increase (P < 0.001). The serum
concentrations of total cholesterol and HDL-cholesterol were
significantly higher in the PCB group than those in C group
(P < 0.001). The addition of taurine to the diet of
PCB-treated rats significantly amplified the increase in total- and
HDL-cholesterol (P < 0.001).
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, P < 0.001). In the case of PCB-treated groups, dietary taurine
significantly amplified the increased amount of cytochrome P-450 caused
by PCB (P < 0.001). The amount of cytochrome b5,
expressed as nmol/g liver, was higher in PCB-treated groups than in
control groups (P < 0.01), but the amount of
cytochrome b5 was unaffected by taurine supplementation.
The hepatic and splenic concentrations of ascorbic acid were
significantly higher in rats fed PCB than those in control rats
(P < 0.01). The addition of taurine to the diets had
no effect on the concentrations of ascorbic acid in the liver and
spleen. Dietary PCB dramatically increased urinary excretion of
ascorbic acid (Fig. 1
, P < 0.001). In the rats that were not treated with
PCB, dietary addition of taurine did not affect the excretion of
ascorbic acid (P > 0.05), whereas in the
PCB-treated rats, dietary taurine significantly amplified the
PCB-induced enhancement of the urinary ascorbic acid excretion all
days of measurements (P < 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-lactone oxidase activity, they must consume
ascorbic acid as a vitamin. The administration of various types of
xenobiotics causes marked increases in urinary excretion and tissue
concentrations of ascorbic acid in rats (Burns et al. 1954
and 1957
, Conney and Burns 1959
, Hollmann and Touster 1962
, Horio and Yoshida 1982
,
Kato et al. 1980
, Lake et al. 1978
,
Longenecker et al. 1940
). Previous reports suggested
that this phenomenon was caused by the enhancement of ascorbic acid
synthesis in the liver, accompanied by the high activities of the
hepatic drug-metabolizing enzymes (Conney and Burns 1959
, Horio et al. 1986
). Xenobiotics induce
some enzymes in the D-glucuronate pathway such as UDP
glucose dehydrogenase, (UDPGT) and ß-glucuronidase (Hollmann and Touster 1962
, Horio and Yoshida 1982
,
Horio et al. 1983
). Among these enzymes, the induction
of UDPGT is thought to be a crucial step for the
xenobiotic-dependent induction of ascorbic acid synthesis
(Horio et al. 1993
). Because the administration of large
amounts of ascorbic acid in the diet ameliorated the toxicity of PCB in
guinea pigs, Kato et al. (1977)
postulated that the
enhancement of ascorbic acid synthesis might be an active adaptive and
protective response to the exposure to PCB. Also in ODS-od/od
(osteogenic disorder Shionogi) rats, which are unable to synthesize
ascorbic acid, the dietary requirement of ascorbic acid for the maximum
induction of hepatic drug-metabolism was increased several-fold
by the administration of PCB (Horio et al. 1986
). In
this study, we examined the effect of dietary taurine on the
concentrations of ascorbic acid and drug-metabolizing enzymes in
rat liver to investigate the possibility that taurine has an adaptive
and protective function in xenobiotic-treated animals. Dietary PCB
significantly enhanced not only the concentrations of ascorbic acid in
urine and the tissues, but also the amounts of cytochromes P-450, which
was consistently accompanied by increases in serum total cholesterol
and HDL-cholesterol. On the other hand, the simultaneous
supplementation of taurine with PCB significantly amplified the urinary
excretion of ascorbic acid and the concentrations of hepatic microsomal
cytochrome P-450 in rats. These results suggested that, in the case of
PCB-treated groups, dietary taurine might enhance the biosynthesis
of ascorbic acid, presumably through the acceleration of the hepatic
drug-metabolizing systems.
Relationships between S-AA and ascorbic acid metabolism have been
investigated. Oda et al. (1986)
reported that the
supplementation of methionine (13.4 or 33.5 mmol/kg diet) or cystine
(as half cystine, 13.4 or 33.5 mmol/kg diet) to a soy protein isolate
(100 g/kg diet) diet containing PCB enhanced the PCB-induced
increase in serum cholesterol and urinary ascorbic acid. Because
S-AA is the first limiting amino acid in soy protein isolate, the
observed effects of S-AA may be due to stimulation of protein
synthesis rather than to a specific action of S-AA. In the present
study, the contents of S-AA in the diets containing 200 g/kg diet
of casein were sufficient, and 30 g/kg diet of taurine also was
supplemented. The excessive dietary supplementation of S-AA
generally is toxic to animals (Benevenga and Harper 1967
). No toxic symptoms due to taurine were observed in this
study, although 30 g/kg diet taurine may be a superphysiological dose
and an excess. Indeed, as a final metabolite of S-AA, taurine is
widely distributed in various tissues in animals, and it is one of the
nonessential amino acids except for in cats. Therefore, it is likely
that the mechanism whereby taurine exerts its effect on ascorbic acid
excretion might be different from that due to methionine or cystine. It
has been suggested that taurine has a function in protecting biological
systems from oxygen, despite its lack of ready oxidizability
(Huxtable 1992
). Hypotaurine and generation of
chlorotaurine are thought to have an antioxidative function in vivo.
Ascorbic acid and taurine might exert antioxidative action additionally
or synergistically. The mechanism by which urinary excretion of
ascorbic acid and hepatic cytochrome P-450 contents were enhanced by
taurine in rats fed PCB is unknown. We are now determining the activity
and gene expression of UDPGT and drug-inducible cytochrome P-450
gene expression. We recently found that cholesterol 7
-hydroxylase,
the rate-limiting step of bile acid synthesis, was induced by
taurine at the mRNA level (Yokogoshi et al. 1999
)
and the hepatic level of apolipoprotein A-I mRNA was increased by
dietary taurine (Mochizuki et al. 1998
, Yokogoshi et al. 1999
). Moreover, PCB-induced malic enzyme gene
expression (Hitomi et al. 1993
) is markedly enhanced by
dietary taurine (Mochizuki et al., unpublished data). Therefore, we
assume that dietary taurine enhances gene expression of enzymes in the
D-glucuronate pathway such as UDPGT and the
drug-inducible cytochromes P-450. Further studies are required to
explore the mechanism of the acceleration of ascorbic acid metabolism
induced by taurine in rats fed a diet containing PCB.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BHT, 2,6-di-tert-2,2-butyl-p-cresol; DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; PCB, polychlorinated biphenyls; S-AA, sulfur-containing amino acids; UDPGT, UDP-glucuronosyltransferase
Manuscript received August 20, 1999. Initial review completed October 11, 1999. Revision accepted December 20, 1999.
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