Melatonin Formation in Pineal Gland from Rats with Hexachlorobenzene Experimental Porphyria

Abstract

Hexachlorobenzene produces an experimental hepatic por-phyria in rats, which is similar to human porphyria cutanea tarda, with hyperpigmentation as one of its characteristic features. Alterations in tryptophan metabolism have been previously observed in this chronic porphyria. Melatonin formation from tryptophan via serotonin shows diurnal rhythmicity in the pineal gland, and higher values are observed during the dark phase of an imposed light-dark cycle. The purpose of this study was to determine the contents of tryptophan and its metabolites in pineal gland of normal and hexachlorobenzene-treated rats in order to find alterations potentially related to porphyria cutanea tarda. Results show that in animals with this experimental porphyria some tryptophan metabolite levels (serotonin and 5-hydroxyindoleacetic acid) increase only during the light period, whereas tryptophan content remained equal to the controls. Hydroxyindole-O-methyltransferase activity also increases by light in pineal gland from hexachlorobenzene-treated rats. On the other hand, tryptophan is converted to melatonin in the dark period, but this route is not exacerbated in hexachloroben-zene porphyria. The relevance of these alterations is discussed in relation to hyperpigmentation, neoplastic and oxidative stress processes associated with this porphyria.

Important neurochemical indoles can be found in the pineal gland. Tryptophan is converted into melatonin in this gland only in the darkness (Reiter and Vaughan 1977; Wurtman and Moskowitz 1977).

In mammals, dietary tryptophan can be metabolized through three different routes as can be seen in Figure 1. One of these routes is its conversion into indole, which is excreted in urine as indoxylsulfate. Other route is the formation of serotonin (5-HT) via 5-hydroxytryptophan (5-HTRP). In a third route, formylkynurenine is formed by the action of tryptophan pyrro-lase (Llambías, Aldonatti, and San Martín de Viale 2003).

Once 5-HT is generated from tryptophan, it can be converted into melatonin (5-methoxy-N-acetyl serotonin) in the pineal gland. It occurs via N-acetylserotonin (NAS) formation. Both 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) derived from 5-HT, but are generated through other routes. Melatonin, the main hormone synthesized in the pineal gland, is secreted at night in all animal species (Arendt and Skene 2005). In pineal glands from most animal species, 5-HIAA and 5-HT contents also have diurnal rhythmicity (Lynch 1971), but the highest values are found at night (or in the dark phase of an imposed light-dark cycle).

Hexachlorobenzene (HCB) produced an experimental hepatic porphyria similar to human porphyria cutanea tarda (PCT) (De Matteis, Prior, and Rimington 1961; San Martín de Viale et al. 1970; Wainstok de Calmanovici et al. 1984). Respect to the effect of HCB on indole pathway it has been reported that dietary HCB is responsible for regional brain concentrations of some tryptophan metabolites and its altered animal behavior in minks and European ferrets (Bleavins et al. 1984). More recently, studies carried out in rats by our group showed significant increases in urinary 5-HT and 5-HIAA and hepatic tryptophan levels in HCB-treated rats. Moreover, 5-HT content in small gut was found to be lower than in controls (Llambías, Aldo-natti, and San Martín de Viale 2003). Some of these alterations would be related to the gluconeogenic blockage observed in experimental PCT (Mazzetti et al. 2004). The lack of signifi-cant variations in 5-HT content found in brain would account for the absence of neurological symptoms in patients with this type of porphyria (Llambías, Aldonatti, and San Martín de Viale 2003).

Melatonin has been considered an antioxidant that prevents injuries resulting from ischemia-reperfusion in rat ileum (Ozacmak et al. 2005). Its protective action against adverse changes is believed to derive from direct free radical scavenging and indirect antioxidant activity, possibly due to its ability to limit free radical generation at the mitochondrial level and to other undefined functions (Reiter et al. 2005). It is worth mentioning that experimental PCT induced by HCB promotes the formation of reactive oxygen species involved in the por-phyrinogenic mechanism elicited by this drug (Billi de Catabbi et al. 1997). On the basis of the already mentioned ability of melatonin to scavenge free radicals, it can be speculated that alterations in the synthesis of this indole compound would be related to the outbreak and development of PCT.

Hyperpigmentation is one of the characteristic features of PCT. With respect to this, it has been reported that 5-HTRP, 5-HT, and some of their catabolites can be converted to melanin, the skin pigment (Costa, Allegri, and De Antoni 1975).

Therefore, it was important to find alterations in trypto-phan metabolism of animals with chronic hepatic porphyria. It was also valuable to establish a relationship between these alterations, hyperpigmentation, and the porphyrinogenic mechanism of this porphyria. This study evaluates the alterations in the formation of melatonin and its intermediates in pineal gland of rats with experimental PCT induced by HCB. To the best of our knowledge, there are no previous reports on the effect of HCB on pineal gland, although Attia et al. (1990) reported that another organochlorine insecticide, lindane, mod-ifies pineal melatonin synthesis in vivo. Thus, for the first time, changes in melatonin, 5-HT, tryptophan, 5-HTOL, NAS, and 5-HIAA levels in pineal from HCB porphyric rats were determined during dark or light period. Likewise, pineal hydroxyindole-O-methyltransferase (HIOMT) activity from normal and HCB-treated rats was measured under light conditions.

MATERIALS AND METHODS

Materials

Rats were fed with Purina Diet 3 (Cabeca, Argentina). Standards used were from Sigma-Aldrich (St. Louis, MO, USA). Methanol was HPLC grade (Sintorgan, Argentina). All the other reagents were analytical grade.

Animal Treatment

Randomly bred female Wistar rats, weighing 160 to 180 g at the beginning of each experiment, were used. All procedures were performed in accordance with international guidelines for the care and use of laboratory animals (Guide for the Care and Use of Laboratory Animals, National Research Council, USA, 1996, and Council of European Communities Directive 86/609/ECC). Rats were kept in a 12-h light/dark cycle, with free access to tap water and food. “Light” rats and “dark” rats were kept from 6 am to 6 pm in the light and in the dark, respectively, and then all were killed at 12 pm. To make them porphyric, rats were given 100 mg HCB/kg body weight by gastric tubing in a single daily dose for 5 days. The drug was suspended in corn oil (12.5 mg/ml). Controls were treated with oil, without HCB, in the same conditions. This protocol caused no deaths, detectable neurological syndromes, or animal suffering (Billi de Catabbi, Aldonatti, and San Martín de Viale 2000). Animals developed a more defined porphyria with this lower HCB dose than with the one used in the continual schedule (Llambías, Aldonatti, and San Martín de Viale 2003; Wainstok de Calmanovici et al. 1984). Then, at least 7 weeks after treatment, rats were killed by decapitation. Pineal glands were immediately removed and kept at − 70°C until use.

Standard Sample Preparation of Tryptophan and Its Metabolites

Stock solutions (10 mg/100 ml) were prepared in filtered bidistilled water with 0.1% ascorbic acid, and then stored at 4°C (Anderson, Young, and Cohen 1982). Diluted standards were prepared daily.

Preparation of Pineal Samples

Pineal glands from normal and porphyric rats were used. Samples were prepared according to Anderson, Young, and Cohen (1982). Frozen pineal glands, stored at − 70°C, were homogenized by sonication for 20 s at 50% with an Ultrasonic Homogenizer 4710 Series (Cole Palmer Instrument, Chicago, IL). Sonication procedures were performed in 0.5 ml ice-cold 0.1 M perchloric acid containing 0.1% ascorbic acid. Samples were centrifuged for 5 min at 10,000 × g. Supernatants were kept at 4°C in the dark until being used the same day. Only the samples used for melatonin determination were kept for 24 h at − 70°C. All samples were filtered through 0.45-μm pore size Millipore filters SJHV013NS (Millipore, Brazil) before being injected.

Apparatus

A Spectra SERIES (Thermo Separation Products, USA) Model P200 liquid chromatograph and a FL2000 fluorescence detector set at excitation and emission wavelengths of 286 and 346 nm, respectively, were used. Samples (50 μl) were injected with a Rheodyne (Cotati, CA) 7125 injector fitted with a 50-μl loop. The separation was carried out on a C₁₈ reverse-phase 3.9 × 300-mm μBondapak column (Waters, Milford, MA) with 10 mM sodium acetate, pH 4.5 and methanol in the ratio 65:35 for melatonin and 85:15 for tryptophan, 5-HT, 5-HTOL, NAS, and 5-HIAA determination, as described by Anderson et al. (1982). Solutions and water were filtered through 0.45-μm fil-ters and degassed by bubbling with helium. The flow rate was 2.0 ml/min.

The column was operated at room temperature and the detector was set on the 0-to-20 range. All chromatograms, retention times, and peak areas were recorded with a Data Jet Integrator (Thermo Separation Products, USA). The integrator was programmed to have an 8 attenuation and a 0.25 cm/min chart speed.

Determination of HIOMT Activity

Pineal HIOMT activity from normal and experimental por-phyric “light” rats was measured according to Itoh, Hattori, and Sumi (1997). Frozen pineal glands, stored at − 70°C, were placed in 1.5-ml polypropylene microcentrifuge tubes on ice, with 0.05 phosphate buffer, pH 7.9. Tissues were homogenized by sonication with a Perkin Elmer 450 Ultrasonic Homogenizer 4710 for three periods of 15 s each. Homogenates were centrifuged at 12000 × g for 10 min at 4°C. Aliquots of 150 μl supernatant were mixed with 150 μl 2 mM N-acetylserotonin plus 0.2 mM S-adenosyl-l-methionine in 0.05 M sodium phosphate buffer, pH 7.9, and then incubated for 1 h at 37°C. Reaction mixtures without N-acetylserotonin or without enzyme were used as controls. The enzymatic reaction was stopped adding 200 μl 3 M perchloric acid, and then centrifuged at 12000 × g for 10 min. The filtered supernatant (100 μl) was analyzed by high performance liquid chromatography (HPLC) according to Itoh, Hattori, and Sumi (1997) using a C₁₈ reverse-phase 3.9 × 300-mm μBondapak column (Waters, Milford, MA). The HPLC mobile phase, consisting of 0.05 M ammonium acetate and 30% methanol, was adjusted to pH 4.3 with acetic acid, and then pumped at a 0.7 ml/min flow rate. The fluorescence detector was set at excitation and emission wavelengths of 280 and 340 nm, respectively.

Quantification

Every peak eluted from the HPLC was confirmed by coelution with the corresponding standard. Peaks were quantified measuring the peak area on the chromatogram. Calibration curves were made under the same conditions described, injecting different amounts of each standard sample in an aqueous solution.

Statistics

The statistical distribution of values obtained was nonpara-metric. Medians and interquartile ranges (IQR) were calculated for each determination. The Mann-Whitney U test was used to compare medians and to determine statistically significant values. For all determinations, p values less or equal to .05 were considered statistically significant.

RESULTS

Porphyria Induction

Animals were treated with HCB according to a noncontinual schedule until they developed a well-defined porphyria. Large amounts of porphyrins were accumulated in liver (400 ± 120 μg/wet g tissue) and excreted by urine (120 ± 20 μg highly carboxylated porphyrins/24 h) 7 to 8 weeks after HCB intoxication ended. Because these biochemical parameters were demonstrated to be effective markers of porphyria induction and HCB persistence, this model can be considered an experimental PCT (Billi de Catabbi, Aldonatti, and San Martín de Viale 2000; Billi de Catabbi et al. 2005; Taira et al. 2004).

HPLC Elution Chromatograms

Figure 2 shows typical HPLC elution diagrams of tryptophan and its metabolites of pineal extracts from normal and porphyric rats killed during light or dark periods. Standard retention times (min) measured with a mobile phase of 10 mM sodium acetate (pH 4.5):methanol were 2.71 (5-HT), 3.75 (tryptophan), 4.98 (5-HIAA), 5.33 (5-HTOL), and 8.31 (NAS) for 85:15 ratio, and 5.26 (melatonin) for 65:35 ratio.

Content of Tryptophan and Its Metabolites in Pineal Gland Obtained during the Light Phase

Table 1 shows medians and interquartile ranges of trypto-phan, 5-HT, and 5-HIAA contents measured in the light phase. Whereas 5-HT and 5-HIAA increased in porphyric materials, tryptophan remained equal to the controls. Melatonin, 5-HTOL, and NAS were not detected in the light phase.

Content of Tryptophan and Its Metabolites in Pineal Gland Obtained during the Dark Phase

Table 2 describes medians and interquartile ranges of tryp-tophan, 5-HT, 5-HIAA, 5-HTOL, NAS, and melatonin contents measured in the dark phase. No changes in the contents of these metabolites were observed in experimental porphyric rats with respect to controls. 5-HTOL was not detected in most of the samples. NAS content measured in normal pineal glands was consistently higher than values previously reported (Anderson, Young, and Cohen 1982).

Determination of HIOMT Activity in Pineal Gland of “Light” Rats

HIOMT was measured in pineal gland from “light” rats to determine its involvement in the increase of 5-HT and 5-HIAA, which are intermediates in the preferred via when melatonin is not formed. A marked increase in pineal-HIOMT activity was observed in “light” HCB-treated rats with respect to normal ones. Medians (expressed in pg/pineal) were 145 for normal and 790 for experimental porphyric rats, whereas IQR values were 411 and 423, respectively.

DISCUSSION

This study demonstrates that the contents of some tryptophan metabolites (5-HT and 5-HIAA) increase only during the light period in rats with HCB experimental porphyria. The fact that pineal HIOMT activity also increases under these conditions suggests that the porphyric state increases the metabolism of tryptophan to 5-HIAA and 5-methoxyindole acetic acid. This last metabolite could not be determined possibly due to its low endogenous concentration. On the other hand, tryptophan is converted into melatonin in the dark period, but its route is not exacerbated in this hepatic porphyria. The changes observed in tryptophan metabolite levels according to illumination in normal and HCB porphyric rats correlate with previous reports on diurnal variations of melatonin and 5-HT contents in rat pineal gland (Lynch, 1971; Snyder et al. 1965). These reports described circadian variations and the influence of environmental illumination on the biochemical activity and composition of the pineal gland. Moreover, Jones and Magnus (1979) found that light might stimulate hepatic porphyria in rats due to a neuroendocrine pathway that includes the pineal gland.

Melatonin has been reported to have antioxidant ability (Reiter et al. 2005), and free radicals formation to play a key role in the HCB porphyrinogenic mechanism (Billi de Catabbi et al. 1997). In fact, reactive oxygen species produced by the uncoupling of microsomal electron transport chain elicited by HCB are involved in the genesis of a heme pathway inhibitor and thus, in the deregulation of heme pathway and porphyria onset characterized by high carboxylated porphyrin accumulation (Wainstok de Calmanovici et al. 1984). Based on this, the results herein obtained, showing that melatonin levels are not altered by HCB, indicate that the enhanced formation of free radicals previously reported in HCB-induced porphyria (Billi de Catabbi et al. 1997) is not due to a defect in melatonin formation. Therefore, this lack of variation in melatonin level would help to trigger the chain of events leading to porphyria induction in which free radicals are involved. This biochemical behavior is different to that observed in human and experimental acute porphyria, in which 5-aminolevulinic acid (ALA) overproduced in liver inhibited pineal melatonin production (Puy, Deybach, and Bogdan 1996). This melatonin decrease would contribute to enhance free radical formation from accumulated ALA (Bechara 1995, Monteiro et al. 1989), with the consequent enzymatic damage, among other injuries. Thus, a self-catalyzing oxidation cycle would be produced. This different expression of melatonin levels in acute (AIP) and chronic (PCT) porphyrias could be related to the fact that hepatic ALA formation and its urinary excretion is lower in rats treated with HCB, a PCT model, than in rats treated with allylisopropyl-acetamide (AIA), an acute porphyria model (Schoenfeld et al. 1991; Wainstok de Calmanovici et al. 1984). It is also worth mentioning that ALA does not alter pineal melatonin levels in fed animals (Daya et al. 1989; Puy, Deybach, and Bogdan 1996), which were the ones used in our study.

HCB not only disturbs tryptophan metabolism, but also alters the heme pathway, as shown by the amount of porphyrins accumulated in the gland. This was determined by spectrophoto-metric and spectrophotofluorometric assays, including their respective spectra. Moreover, characteristic porphyrin peaks were observed at the Soret band in ultraviolet (UV)-visible spectrum, and two fluorescence emission peaks were measured in the 590-to 650-nm zone in the fluorescence emission spectrum when samples were excited at 403 nm (data not shown). The dose of HCB used in our study was able to induce PCT-type porphyria in accordance with previous reports (Krishnan, Brodeur, and Charbonneau 1991).

It can be considered that HCB enters the pineal gland because (a) lipids are present in the composition of this gland (Huxley and Tapp 1972), (b) HCB is a highly lipophilic chemical which is incorporated in fatty acid chains of membrane lipids (Koszo et al. 1982), and (c) HCB penetrates into different tissues and accumulates in many organs (Agency for Toxic Substances and Disease Register 2002). Once HCB is within the pineal gland, it increases the biosynthesis and utilization of 5-HT and 5-HIAA under light conditions, because enhanced levels of these metabolites as well as increased enzymatic activity of HIOMT are here found.

To determine the relevance of these increases, some considerations must be taken into account. On the one hand, increased urinary and serum 5-HIAA levels are used in humans as markers of primary hepatic carcinoid tumors (Iwao et al. 2001) and of several other kind of tumors and their metastases (Fiorentini et al. 2004, Zuetenhorst et al. 2003). Thus, because 5-HIAA has been associated with cancer, its increase not only in pineal gland, but also in HCB porphyric rat urine (Llambías, Aldonatti, and San Martín de Viale 2003), would suggest that a neoplastic process could be related to this porphyria. In fact, HCB carcinogenic activity in different animals has been demonstrated by the manifestation of cancer in different organs (Cabral and Shubik 1986; Plante et al. 2002).

On the other hand, the relationship between tryptophan metabolism and pigmentation is a complex one, and it was demonstrated that 5-HTRP, 5-HT, and some of their catabo-lites can form melanin (Foldes et al. 1988). Therefore, increases in 5-HT levels observed in our study and in a previous report (Llambías, Aldonatti, and San Martín de Viale 2003) would contribute to enhance melanin circulating pool. This would be relevant for the hyperpigmentation process, a characteristic feature of human PCT, because Costa, Allegri, and De Antoni (1975) reported that these two tryptophan metabolites and some of their catabolites can be converted into melanin, the skin pigment.