Metabolic Brain Disease, Vol. 7, No. 4, 1992
The Effect of A f l a t o x i n B1 Exposure on Serotonin Metabolism: Response to a Tryptophan Load T.D. Kimbroughl'2,G.C. Llewellyn 3 and L.B. Weekley 4 Received September 14, 1992; accepted December 7, 1992 Semi-chronic exposure of ICR male Mice to Aflatoxin BI (AFB1) in non-toxic doses decreased brain serotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and catecholamines without altering tryptophan (TRP) or tyrosine (TYR) levels. A TRP load (300 mg/kg, i.p. x 2 hours) slightly increased brain TRP levels while causing a slight decrease in 5-HT and 5-HIAA in control animals. A TRP load in AFB 1 treated mice increased brain 5-HT and 5-HIAA. The TRP load caused a further reduction in brain catecholarnines without altering TYR levels. Exposure to AFB significantly increased lung TRP levels without altering 5-HT or 5H/AA levels. TRP loading increased lung TRP concentrations in control mice. However, in AFB1 treated mice the increase was not significantly elevated above the level caused by AFB1 treatment alone. Lung 5-HT or 5-HIAA levels in control or AFB1 treated mice are not significantly altered by TRP loading. These experiments demonstrate that AFB1 alters brain and lung TRP metabolism. KEY WORDS: Aflatoxin B1; brain; lung; serotonin; tryptophan. INTRODUCTION Aflatoxin B1 (AFB1), a toxic fungal metabolite produced by Aspergillus and Penicillin species of fungi, is a low level contaminant of human and animal food supplies (Connaughton, 1989; Ezzell, 1988; Jelinek et al., 1989; Pensala et al., 1977). Aflatoxin B1, a k n o w n hepatotoxin and neurotoxin ( I k e g w u o n o , 1983) has been associated epidemiologically with several pathological conditions including cancers of the liver (Adamson et al.., 1976), encephalopathy and fatty degeneration of the viscera (Reye's Syndrome) (Becroft et al., 1972; Burguera, 1986; Chaves-Carballo, 1976; Dvorackova et al., 1977 Ryan et al., 1979; Shank et al., 1971). Previous studies have shown tryptophan (TRP) metabolism is altered following AFT treatment (Coulombe and Sharrna, 1985; 1 Department of Biological Sciences, Box 2012, Virginia Commonwealth University, Richmond, VA 23284. 2 To whom correspondence should be addressed. 3 2107 Dresden Rd., Richmond, VA 23284. 4 Department of Biomedical Sciences, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
175 0885-7490/92/1200-0175506.50,t0 9 1992 PlenumPublishing Corporation
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Weekley et aI, 1989i Weeldey, 1991). This study was undertaken to examine the response to a TRP load in the brain and lung of AFBI exposed mice.
MATERIALS AND METHODS Animals and Drug Treatment Adult male ICR mice (31-43 grams) were acclimated to their cages (two mice per cage), diet (laboratory meal for rats, hamsters and mice, Ralston Purina Co., Inc., st. Louis, MO), and photoperiod (12:12 L:D with lights on from 0800 to 2000 hours) for two weeks prior to initiation of these experiments. Food and tap water was offered ad libitvm throughout the experiment. Following the acclimation period mice were given an injection of AFB1 (Calbiochem, LaJolla, CA.) (10 Ixg/kg i.p.) or vehicle (1% acetone in 0.9%NaC1, 10 I.d/gram body weight) once every 72 hours for a period of 21 days. Seventy hours following the last injection of AFBI (or vehicle) mice were injected with L-tryptophan t300 mg/kg, i.p.) or saline and killed 2 hours later. Indoleamine and Catecholamine Assays The brainstem and lungs were removed from the freshly killed animal and homogenized in 2.0 ml of acidified-n-butanol. Following centrifugation (4,000 g x 15 min at 4~ 1.5 ml of the supernatant was returned to a second set of tubes containing 4.0 ml of n-heptane and 0.3 ml of 0.01N HCI. Tryptophan, tyrosine, serotonin, and catecholamines were extracted into the acid layer (Maickel et al., 1968). The organic layer was washed a second time with 0.5 ml of 0.033M NaHCO3 to extract 5-hydroxyindole-3-acetic acid (5-HIAA). Serotonin extracted into the 0.1 N HC1 was assayed fluorometrically following reaction with 0-phthalaldehyde (OPT) as previously described (Ansell and Beeson, 1968) and quantitated. 5-Hydroxyindole-3-acetic acid (5-HIAA) was extracted into the 0.033 M NaHCO3 and various levels were assayed fluorometrically (360 nm excitation wavelength and 480 nm emission wavelength) following reaction with 0-phthalaldehyde (OPT). For determination of tissue tryptophan levels, 0.2 ml of tissue extract was deproteinized by adding it to 10% lrichloroacetic acid and then centfifugating' for 10 minutes at 15,000 X g. The supernatant (0.1 ml) was removed, treated with 3 x 10 -4 M FeC13 in 10% trichloroacetic acid and subsequently added to 0.1 ml of 2% formaldehyde(Denckla and Dewey, 1967; Bloxam and Warren, 1974). The tubes were stoppered and placed in a boiling water bath for one hour. Trichloroacetic acid (10%) was used to replenish lost volume and tryptophan levels were quantified fluorometrically (373 nm excitation wavelength and 452 nm emission wavelength). Following extracting in 0.01N HC1, tyrosine was quantitated by deproteinizing with 0.1 ml of 30% trichloroacetic acid. Following a 10 minute centrifugation (15,000 X g), the supernatant (0.2 ml) was added to 1.0 ml of 0.1% 1-nitroso-2-napthol in 95% ethanol and 1.0 ml of 20% NAN02 in 4.0 M HNO3. The tube was stoppered, shaken, and incubated in a water bath at 55~ for 30 minutes. After cooling, the unreacted 1-nitroso-2-napthol was
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Fig 1. Brain concentrations of TRP, 5-HT, or 5-HIAA following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB1 treatments. Control levels of TRP, 5-HT, and 5-HIAA were determined to be 228.2 + 5.7 ~g/g, 74.8 + 4.3 ng/g, and 576.1 + 44.9 ng]g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n--A). * significantly different from the control treatment group # significantly different from the AF81 treatment group extracted by washing with 5.0 ml of ethylene dichloride. Tyrosine levels were determined fluorometrically (460 nm excitation and 570 nm emission)(Waalkes and Undenfriend, 1957). Total catecholamine levels in the 0.01 N HCI tissue extract were determined fluorometricaUy using the trihydroxyindole method (Chang, 1964). To 0.1 ml of the 0.01N HC1 extract was added 0.2 ml of 0.1 M ethylenediaminetetraacetic acid (EDTA) in 1 M sodium acetate followed by 0.1 ml of 0.1 N iodine in absolute ethanol. After exactly 2 minutes 0.2 ml of 0.1 N sodium sulfite in 5 N sodium hydroxide was added and following another 2 min period 0.2 ml of 0.1 N sodium sulfite in 5N sodium hydroxide was added. Two min later, 0.1 ml o f l N acetic was added. To determine catecholamine levels the extract was placed in a boiling water bath for 5 min and, following sample cooling, fluorescence determined (385 nm excitation wavelength and 485 nm emission wavelength).
Statistical Analysis All Tissue amine and amino acid levels were corrected for recovery. Individual comparisons were made with a one-tailed students' t-test. Values with a p>0.05 were considered significant.
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Fig 2. Brain concentrations of tyrosine and catecholamines following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB1 treatments. Control levels of tyrosine and catecholamines were determined to be 41.8 + 1.9 I.tg/g and 235.7 + 14.9 ng/g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n=4). # significantly different from the control treatment group # significantly different from the AFB 1 treatment group
RESULTS Brain
Brain TRP levels are not altered by AFB1 treatment while 5-HT and 5-HIAA levels are significantly reduced (Fig 1). A TRP load does not significantly alter brain TRP levels while brain 5-HT and 5-HIAA levels are slightly reduced. In AFBI treated mice, a TRP load increases brain 5-HT levels without altering TRP or 5-HIAA levels. Brain TYR levels are not altered by AFB1 treatment or a TRP load (Fig 2). Brain catecholamines are reduced by AFB1 treatment and further reduced by TRP loading of AFBI treated mice.
Lung Aflatoxin B 1 treatment significantly increased lung TRP levels. The elevation in lung TRP caused by AFB1 treatment is not significantly altered by AFBI treatment or TRP loading. However, TRP loading alone also increases the TRP levels in the lung (Fig 3).
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Fig 3. Lung concentration of TRP, 5-HT, and 5-HIAA following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB 1 treatments. Control levels of TRP, 5-HT, and 5-HIAA were determined to be 2.80 + 0.48 ~tg/g, 7.78 + 2.51ng/g, and 189.1 + 28.7 ng/g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n-4). # significantly different from the control treatment group. * significantly different from the TRP load treatment group DISCUSSION This concentration of AFB1 is subtoxic as assessed by the lack of effect on body weight and hematocrit (Weekley, 1991) and an absence of gross clinical signs of toxicity. Mice were used in these studies since they are somewhat resistant to aflatoxin induced toxicity (Platnow, 1964). The AFB1 LDs0 in mice is approximately 9 mg/kg and the mice received a cumulative dose of 70 ug/kg or about 0.78 percent of the LD50 over a 21 day period. Aflatoxin B1 treatment p e r se caused a significant elevation in lung tryptophan levels while lung 5-HT and 5-HIAA levels are only slightly increased. The reason for this increase in lung tryptophan following AFBI treatment is not clear. However, it does appear to be tissue specific in that AFB1 did not alter TRP levels in brain. The lack of effect on lung 5-HT and 5-HIAA suggests that TRP may be metabolized via alternate pathways. Mouse indoleamine 2,3-dioxygenase is found in brain and lungs and has a high affinity for tryptophan (Km = 20 lxM), suggesting that it actively oxidizes tryptophan. An alternate explanation is that any excess 5-HT or 5HIAA synthesized from TRP is metabolized via indoleamine 2,3 dioxygenase. Indoleamine 2,3 dioxygenase utilizes superoxide anion, TRP and related indoles as substrates. AFB1 did not alter brain TRP levels while 5-HT and 5-HIAA levels are significantly decreased, an effect consistent with previous observations (Weekley et al., 1978; Weekley et al., 1985; Weekley, 1991). Surprisingly, TRP loading only slightly increased brain TRP levels while 5-HT and 5-HIAA levels are slightly
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decreased in controls. This failure of a TRP load to increase brain TRP is not clear although it may be a species or strain difference (Fernstrom and Wurtman, 1973). The increase in lung TRP by a TRP load indicates the animal had an increase in circulating TRP from the injection and suggests that the mice did not transport TRP into brain or that it was rapidly metabolized to other products. Indeed, Gal and Sherman (1978) reported that about induced toxicity (Plamow, 1964). The AFB1LDso in mice is approximately 9 mg/Kg and the mice received a cumulative dose of 70 ug/kg or about 0.78 percent of the LD50 over a 21 day period. Aflatoxin B1 treatment p e r se caused a significant elevation in lung tryptophan levels while lung 5-HT and 5-HIAA levels are only slightly increased. The reason for this increase in lung tryptophan following AFBI treatment is not clear. However, it does appear to be tissue specific in that AFB1 did not alter TRP levels in brain. The lack of effect on lung 5-HT and 5-HIAA suggests that TRP may be metabolized via alternate pathways. Mouse indoleamine 2,3-dioxygenase is found in brain and lungs and has a high affinity for tryptophan (Km = 20 tiM), suggesting that it actively oxidizes tryptophan. An alternate explanation is that any excess 5-HT or 5HIAA synthesized from TRP is metabolized via indoleamlne 2,3 dioxygenase. Indoleamine 2,3 dioxygenase utilized superoxide anion, TRP and related indoles as substrates. AFBI did not alter brain TRP levels while 5-HT and 5-HIAA levels are significantly decreased, an effect consistent with previous observations (Weekley et al., 1978; Weekley et al., 1985; Weekley, 1991). Surprisingly, TRP loading only slightly increased brain TRP levels while 5-HT and 5-HIAA levels are slightly decreased in controls. This failure of a TRP load to increase brain TRP is not clear although it may be a species or strain difference (Fernstrom and Wunman, 1973). The increase in lung TRP by a TRP load indicates the animal had an increase in circulating TRP from the injection and suggests that the mice did not transport TRP into brain or that it was rapidly metabolized to other products. Indeed, Gal and Sherman (1978) reported that about 40 percent of the cerebral pool of kynurenine is formed by indoleamine 2,3 dioxygenase while the rest is taken up from the circulation. Indeed, both bacterial lipopolysaccharide and a viral infection have been shown to activate indole 2,3 dioxygenase possibly indirectly via their release of interferon (Sayama et al., 1981). Changes in the activity of indole 2,3 dioxygenase followinq AFT treatment would alter CNS and circulating levels of metabolites such as kynurenine and quinolinic acid, both of which have been implicated as neurodegenerative agents (EI-Defrawy et al., 1986). These shifts in central and systemic tryptophan metabolism following AFT treatment combined with a decrease in hepatic carbamyl phosphate synthetase (Ahmed and Singh, 1984) and ornithine transcarbamylase (Ramachandra et al., 1975) with the resulting hyperammonemia may play a key role in the neurotoxicity of AFT. Ikegwuovo (1983) has reported that AFBI causes enzymatic changes in brain consistent with neuronal degeneration. Indeed, a possible link between AFBI and Reyes Syndrome, a disease characterized by neuronal degeneration has been suggested (Ryan et al., 1979). These results and those from previous investigations indicate that AFBI causes perturbations in amino acid metabolism. Such alteration may contribute to the development of Reye's syndrome. However, Reye's syndrome is a multifactorial disease and any single factor cannot be clearly identified as a causative agent.
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REFERENCES
Adamson,R.H., Correa, P., Sieber, S.M., McIntire, K. and Dalgard, D. (1976). Carcinogenicity of aflatoxin 81 in Rhesus monkeys: two additional cases of primary liver cancer. J. Natl. Cancer Inst. 57: 67-68. Ahmed, N. and Singh, U.W. (1984). Effect of aflatoxin B1 on brain serotonin and catecholamines in chickens. Toxicol. Lett. 21: 365-367. Anseli, G., and Beeson, M.(1968). A rapid and sensitive procedure for the combined assay of noradrenaline, dopamine and serotonin in a single brain sample. Anal B/ochem. 23: 196206. Becroft, D. and Webster, D.R. (1972). Aflatoxirts and Reye's disease. Br. Med. J. 4:117-119. Bloxam, D. and warren, W.(1974). Error in the determination of tryptophan by the method of Denckla and Dewey. A revised procedure. Analyt. Biochem. 60: 621-624. Burguera, J.A. (1986). Presence of aflatoxin Bt in human liver referred to as Reye's syndrome in Venezuela. Acta Cient. Vuenz. 37(3): 325-326. Chang, C.C. (1964). Fluoromelric determination of catecholamines Intemat. J. Neuropharmacol. 3: 643-646. Chaves-CarbaUo, E., Ellefson, R.D. and Gomez, M.R. (1976). Aflatoxin in the liver of a patient with Reye-Johnson syndrome. Mayo Clin. Proc. 51: 48-52. Cormaughton, D. (1989). The threat of aflatoxins. J. Am. Vet. Med. Assoc. 194(6): 743-746. Coulombe, R.A., and Sharma, R.P. (1985). Effect of repeated dietary exposure of AFB1 on brain biogenic amines and metabolites in the rat. Toxicol. and Appl. Pharmacol. 80: 496501. Denclda, W.D., and Dewey, H.K.(1967). The determination of tryptophan in plasma, liver and urine. J. Lab. Clin.Med. 69: 160-169. Dvorackova, I., Kusak, V. and Vesely, D. (1977). Aflatoxin and encephalopathy with fatty degeneration of viscera (Reye). Ann. Nutr. Aliment. 31: 977-987. E1-Defrawy, S.R., Boegman, R.J., Jhamandos, K. and Bennigen, R.J.(1986). The neurotoxic action of quinollnic acid in the CNS. Canad. J. Physiol. Pharmacol. 64(3): 369-375. Ezzell, C. (1988). Aflatoxin contamination of US com. Nature 335 (6193): 757. Femstrom, J.D., and Wurtman, R.J.(1973 ). Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178: 414-416. Gal, E. M. and Sherman, A.D.(1978). Synthesis and metabolism of L-kynurenine in rat brain. J. Neurochem. 30: 607-614. Ikegwuovo, F.I. (1983). The neurotoxicity of aflatoxin BI in the rat. Toxicol. 28:247-259 Jelinek, C.F. Pohland, A.E., Wood, G.E.(1989). Worldwide occurrence of mycotoxins in foods and feeds- an update. J. Assoc. Off. Analyst. Chem. 72 (2): 223-230. Pensala, O., Niskanen, A. and Lahfinen, S. (1977). The occurrence of aflatoxin in nuts and nut products imported to Finland for human consumption during the years 1974-1976. Nord. Vet. Med. 29(7-8): 347-355. Plator~ow, N. (1964). Toxicity of aflatoxins in the mouse. Vet. Record. 76: 589-590. Ramachandra, P.M., Jayonthi, B.N., Venkitasubramonian, T.A.(1975). Effect of aflatoxin on oxidative phosphorylation by rat liver mitochondria. Chem. Biol. Interact. 10(2): 123-131. Ryan, N.J., Hogan, G.R., Hayes, A.W., Unges, P.D. and Siraj, M.Y.(1979). Aflatoxin BI: its role in the etiology of Reye's syndrome. Pediatrics 64:71-74. Sayama, S., Yoshida, R., Oku, T., Imanishi, J., Kishida, T. and Hayaishi, 0.(1981). Inhibition of interferon mediated induction of indoleamine 2, 3 dioxygenaqe in mouse lung by inhibition of prostaglandins. Proc. Natl. Acad. Sci. USA. 78: 7327-7330. Shank, R.C., Bourgeois, C.H., Keschamaras, N. et al. (1971). Aflatoxins in autopsy specimens from Thai children with an acute disease of unknown aetislogy. Food Cosmet. Toxicol. 9: 501-506.
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Takikawa, O., Yoshida, R., Kido, R., Hayaishi, O. (1986). Tryptophan degradation in mice initiated by indoleamine 2, 3 dioxygenase. J. Biol. Chem. 261(8): 3648- 3653. Waalkes, T.P., and Undenfxiend, S. (1957). A fluorometric method for the estimation of tyrosine ha plasma and tissues. J. Lab. Clin. Med. 50: 773-736. Weeldey, L.B. (1991). Aflatoxin B1 alters central and systemic tryptophan and tyrosine metabolism: Influence of immunomodulatory drugs. Metab, Brain Dis. 6(1): 19-32, Weekley, L.B., Kimbrough, T.D., and Llewellyn, G.C. (1985). Disturbances in tryptophan metabolism in rats following chronic dietary aflatoxin treatment. Drug Chem. Toxicol. 8: 145-154. Weekley, L.B., Morgan, J.D., Rea, F.W., Kimbrough, T.D., and Llewellyn, G.C. (1978). Alterations of brain and intestine serotonin levels in hamsters pretreated with dietary aflatoxin. Cancer Lea. 5: 75-80. Weekley, L.B., O'Rear, C.E., Kimbrough, T.D., and LleweUyn, G.C. (1989). Differential changes in rat brain tryptophan serotonin and tyrosine levels following acute AFB1 treatment. Toxicol. Lett. 47: 173-177.
The Effect of A f l a t o x i n B1 Exposure on Serotonin Metabolism: Response to a Tryptophan Load T.D. Kimbroughl'2,G.C. Llewellyn 3 and L.B. Weekley 4 Received September 14, 1992; accepted December 7, 1992 Semi-chronic exposure of ICR male Mice to Aflatoxin BI (AFB1) in non-toxic doses decreased brain serotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and catecholamines without altering tryptophan (TRP) or tyrosine (TYR) levels. A TRP load (300 mg/kg, i.p. x 2 hours) slightly increased brain TRP levels while causing a slight decrease in 5-HT and 5-HIAA in control animals. A TRP load in AFB 1 treated mice increased brain 5-HT and 5-HIAA. The TRP load caused a further reduction in brain catecholarnines without altering TYR levels. Exposure to AFB significantly increased lung TRP levels without altering 5-HT or 5H/AA levels. TRP loading increased lung TRP concentrations in control mice. However, in AFB1 treated mice the increase was not significantly elevated above the level caused by AFB1 treatment alone. Lung 5-HT or 5-HIAA levels in control or AFB1 treated mice are not significantly altered by TRP loading. These experiments demonstrate that AFB1 alters brain and lung TRP metabolism. KEY WORDS: Aflatoxin B1; brain; lung; serotonin; tryptophan. INTRODUCTION Aflatoxin B1 (AFB1), a toxic fungal metabolite produced by Aspergillus and Penicillin species of fungi, is a low level contaminant of human and animal food supplies (Connaughton, 1989; Ezzell, 1988; Jelinek et al., 1989; Pensala et al., 1977). Aflatoxin B1, a k n o w n hepatotoxin and neurotoxin ( I k e g w u o n o , 1983) has been associated epidemiologically with several pathological conditions including cancers of the liver (Adamson et al.., 1976), encephalopathy and fatty degeneration of the viscera (Reye's Syndrome) (Becroft et al., 1972; Burguera, 1986; Chaves-Carballo, 1976; Dvorackova et al., 1977 Ryan et al., 1979; Shank et al., 1971). Previous studies have shown tryptophan (TRP) metabolism is altered following AFT treatment (Coulombe and Sharrna, 1985; 1 Department of Biological Sciences, Box 2012, Virginia Commonwealth University, Richmond, VA 23284. 2 To whom correspondence should be addressed. 3 2107 Dresden Rd., Richmond, VA 23284. 4 Department of Biomedical Sciences, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
175 0885-7490/92/1200-0175506.50,t0 9 1992 PlenumPublishing Corporation
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Weekley et aI, 1989i Weeldey, 1991). This study was undertaken to examine the response to a TRP load in the brain and lung of AFBI exposed mice.
MATERIALS AND METHODS Animals and Drug Treatment Adult male ICR mice (31-43 grams) were acclimated to their cages (two mice per cage), diet (laboratory meal for rats, hamsters and mice, Ralston Purina Co., Inc., st. Louis, MO), and photoperiod (12:12 L:D with lights on from 0800 to 2000 hours) for two weeks prior to initiation of these experiments. Food and tap water was offered ad libitvm throughout the experiment. Following the acclimation period mice were given an injection of AFB1 (Calbiochem, LaJolla, CA.) (10 Ixg/kg i.p.) or vehicle (1% acetone in 0.9%NaC1, 10 I.d/gram body weight) once every 72 hours for a period of 21 days. Seventy hours following the last injection of AFBI (or vehicle) mice were injected with L-tryptophan t300 mg/kg, i.p.) or saline and killed 2 hours later. Indoleamine and Catecholamine Assays The brainstem and lungs were removed from the freshly killed animal and homogenized in 2.0 ml of acidified-n-butanol. Following centrifugation (4,000 g x 15 min at 4~ 1.5 ml of the supernatant was returned to a second set of tubes containing 4.0 ml of n-heptane and 0.3 ml of 0.01N HCI. Tryptophan, tyrosine, serotonin, and catecholamines were extracted into the acid layer (Maickel et al., 1968). The organic layer was washed a second time with 0.5 ml of 0.033M NaHCO3 to extract 5-hydroxyindole-3-acetic acid (5-HIAA). Serotonin extracted into the 0.1 N HC1 was assayed fluorometrically following reaction with 0-phthalaldehyde (OPT) as previously described (Ansell and Beeson, 1968) and quantitated. 5-Hydroxyindole-3-acetic acid (5-HIAA) was extracted into the 0.033 M NaHCO3 and various levels were assayed fluorometrically (360 nm excitation wavelength and 480 nm emission wavelength) following reaction with 0-phthalaldehyde (OPT). For determination of tissue tryptophan levels, 0.2 ml of tissue extract was deproteinized by adding it to 10% lrichloroacetic acid and then centfifugating' for 10 minutes at 15,000 X g. The supernatant (0.1 ml) was removed, treated with 3 x 10 -4 M FeC13 in 10% trichloroacetic acid and subsequently added to 0.1 ml of 2% formaldehyde(Denckla and Dewey, 1967; Bloxam and Warren, 1974). The tubes were stoppered and placed in a boiling water bath for one hour. Trichloroacetic acid (10%) was used to replenish lost volume and tryptophan levels were quantified fluorometrically (373 nm excitation wavelength and 452 nm emission wavelength). Following extracting in 0.01N HC1, tyrosine was quantitated by deproteinizing with 0.1 ml of 30% trichloroacetic acid. Following a 10 minute centrifugation (15,000 X g), the supernatant (0.2 ml) was added to 1.0 ml of 0.1% 1-nitroso-2-napthol in 95% ethanol and 1.0 ml of 20% NAN02 in 4.0 M HNO3. The tube was stoppered, shaken, and incubated in a water bath at 55~ for 30 minutes. After cooling, the unreacted 1-nitroso-2-napthol was
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Fig 1. Brain concentrations of TRP, 5-HT, or 5-HIAA following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB1 treatments. Control levels of TRP, 5-HT, and 5-HIAA were determined to be 228.2 + 5.7 ~g/g, 74.8 + 4.3 ng/g, and 576.1 + 44.9 ng]g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n--A). * significantly different from the control treatment group # significantly different from the AF81 treatment group extracted by washing with 5.0 ml of ethylene dichloride. Tyrosine levels were determined fluorometrically (460 nm excitation and 570 nm emission)(Waalkes and Undenfriend, 1957). Total catecholamine levels in the 0.01 N HCI tissue extract were determined fluorometricaUy using the trihydroxyindole method (Chang, 1964). To 0.1 ml of the 0.01N HC1 extract was added 0.2 ml of 0.1 M ethylenediaminetetraacetic acid (EDTA) in 1 M sodium acetate followed by 0.1 ml of 0.1 N iodine in absolute ethanol. After exactly 2 minutes 0.2 ml of 0.1 N sodium sulfite in 5 N sodium hydroxide was added and following another 2 min period 0.2 ml of 0.1 N sodium sulfite in 5N sodium hydroxide was added. Two min later, 0.1 ml o f l N acetic was added. To determine catecholamine levels the extract was placed in a boiling water bath for 5 min and, following sample cooling, fluorescence determined (385 nm excitation wavelength and 485 nm emission wavelength).
Statistical Analysis All Tissue amine and amino acid levels were corrected for recovery. Individual comparisons were made with a one-tailed students' t-test. Values with a p>0.05 were considered significant.
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Fig 2. Brain concentrations of tyrosine and catecholamines following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB1 treatments. Control levels of tyrosine and catecholamines were determined to be 41.8 + 1.9 I.tg/g and 235.7 + 14.9 ng/g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n=4). # significantly different from the control treatment group # significantly different from the AFB 1 treatment group
RESULTS Brain
Brain TRP levels are not altered by AFB1 treatment while 5-HT and 5-HIAA levels are significantly reduced (Fig 1). A TRP load does not significantly alter brain TRP levels while brain 5-HT and 5-HIAA levels are slightly reduced. In AFBI treated mice, a TRP load increases brain 5-HT levels without altering TRP or 5-HIAA levels. Brain TYR levels are not altered by AFB1 treatment or a TRP load (Fig 2). Brain catecholamines are reduced by AFB1 treatment and further reduced by TRP loading of AFBI treated mice.
Lung Aflatoxin B 1 treatment significantly increased lung TRP levels. The elevation in lung TRP caused by AFB1 treatment is not significantly altered by AFBI treatment or TRP loading. However, TRP loading alone also increases the TRP levels in the lung (Fig 3).
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Fig 3. Lung concentration of TRP, 5-HT, and 5-HIAA following sham or AFB1 injections. In some experiments, animals were given TRP loads following sham or AFB 1 treatments. Control levels of TRP, 5-HT, and 5-HIAA were determined to be 2.80 + 0.48 ~tg/g, 7.78 + 2.51ng/g, and 189.1 + 28.7 ng/g, respectively. Each bar is presented as the percent of control values and expressed as the mean + S.E.M. (n-4). # significantly different from the control treatment group. * significantly different from the TRP load treatment group DISCUSSION This concentration of AFB1 is subtoxic as assessed by the lack of effect on body weight and hematocrit (Weekley, 1991) and an absence of gross clinical signs of toxicity. Mice were used in these studies since they are somewhat resistant to aflatoxin induced toxicity (Platnow, 1964). The AFB1 LDs0 in mice is approximately 9 mg/kg and the mice received a cumulative dose of 70 ug/kg or about 0.78 percent of the LD50 over a 21 day period. Aflatoxin B1 treatment p e r se caused a significant elevation in lung tryptophan levels while lung 5-HT and 5-HIAA levels are only slightly increased. The reason for this increase in lung tryptophan following AFBI treatment is not clear. However, it does appear to be tissue specific in that AFB1 did not alter TRP levels in brain. The lack of effect on lung 5-HT and 5-HIAA suggests that TRP may be metabolized via alternate pathways. Mouse indoleamine 2,3-dioxygenase is found in brain and lungs and has a high affinity for tryptophan (Km = 20 lxM), suggesting that it actively oxidizes tryptophan. An alternate explanation is that any excess 5-HT or 5HIAA synthesized from TRP is metabolized via indoleamine 2,3 dioxygenase. Indoleamine 2,3 dioxygenase utilizes superoxide anion, TRP and related indoles as substrates. AFB1 did not alter brain TRP levels while 5-HT and 5-HIAA levels are significantly decreased, an effect consistent with previous observations (Weekley et al., 1978; Weekley et al., 1985; Weekley, 1991). Surprisingly, TRP loading only slightly increased brain TRP levels while 5-HT and 5-HIAA levels are slightly
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decreased in controls. This failure of a TRP load to increase brain TRP is not clear although it may be a species or strain difference (Fernstrom and Wurtman, 1973). The increase in lung TRP by a TRP load indicates the animal had an increase in circulating TRP from the injection and suggests that the mice did not transport TRP into brain or that it was rapidly metabolized to other products. Indeed, Gal and Sherman (1978) reported that about induced toxicity (Plamow, 1964). The AFB1LDso in mice is approximately 9 mg/Kg and the mice received a cumulative dose of 70 ug/kg or about 0.78 percent of the LD50 over a 21 day period. Aflatoxin B1 treatment p e r se caused a significant elevation in lung tryptophan levels while lung 5-HT and 5-HIAA levels are only slightly increased. The reason for this increase in lung tryptophan following AFBI treatment is not clear. However, it does appear to be tissue specific in that AFB1 did not alter TRP levels in brain. The lack of effect on lung 5-HT and 5-HIAA suggests that TRP may be metabolized via alternate pathways. Mouse indoleamine 2,3-dioxygenase is found in brain and lungs and has a high affinity for tryptophan (Km = 20 tiM), suggesting that it actively oxidizes tryptophan. An alternate explanation is that any excess 5-HT or 5HIAA synthesized from TRP is metabolized via indoleamlne 2,3 dioxygenase. Indoleamine 2,3 dioxygenase utilized superoxide anion, TRP and related indoles as substrates. AFBI did not alter brain TRP levels while 5-HT and 5-HIAA levels are significantly decreased, an effect consistent with previous observations (Weekley et al., 1978; Weekley et al., 1985; Weekley, 1991). Surprisingly, TRP loading only slightly increased brain TRP levels while 5-HT and 5-HIAA levels are slightly decreased in controls. This failure of a TRP load to increase brain TRP is not clear although it may be a species or strain difference (Fernstrom and Wunman, 1973). The increase in lung TRP by a TRP load indicates the animal had an increase in circulating TRP from the injection and suggests that the mice did not transport TRP into brain or that it was rapidly metabolized to other products. Indeed, Gal and Sherman (1978) reported that about 40 percent of the cerebral pool of kynurenine is formed by indoleamine 2,3 dioxygenase while the rest is taken up from the circulation. Indeed, both bacterial lipopolysaccharide and a viral infection have been shown to activate indole 2,3 dioxygenase possibly indirectly via their release of interferon (Sayama et al., 1981). Changes in the activity of indole 2,3 dioxygenase followinq AFT treatment would alter CNS and circulating levels of metabolites such as kynurenine and quinolinic acid, both of which have been implicated as neurodegenerative agents (EI-Defrawy et al., 1986). These shifts in central and systemic tryptophan metabolism following AFT treatment combined with a decrease in hepatic carbamyl phosphate synthetase (Ahmed and Singh, 1984) and ornithine transcarbamylase (Ramachandra et al., 1975) with the resulting hyperammonemia may play a key role in the neurotoxicity of AFT. Ikegwuovo (1983) has reported that AFBI causes enzymatic changes in brain consistent with neuronal degeneration. Indeed, a possible link between AFBI and Reyes Syndrome, a disease characterized by neuronal degeneration has been suggested (Ryan et al., 1979). These results and those from previous investigations indicate that AFBI causes perturbations in amino acid metabolism. Such alteration may contribute to the development of Reye's syndrome. However, Reye's syndrome is a multifactorial disease and any single factor cannot be clearly identified as a causative agent.
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