2021 Volume 46 Issue 8 Pages 379-389
Activated charcoal (AC) is a potential candidate antidote against dioxins. However, it is difficult to take AC as a supplement on a daily basis, because its long-term ingestion causes side effects such as constipation and deficiency of fat-soluble essential nutrients and hypocholesterolemia. Alginate-coated AC, termed Health Carbon (HC), was developed to decrease the side effects of AC, but its pharmacological effects, including side effects, remains unclear. Here, we show that HC enhanced fecal excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and decreased some side effects of unmodified AC, such as hypocholesterolemia, in male mice. Basal diet mixed with HC or unmodified AC at various concentrations was fed to mice for 16 days following a single intraperitoneal administration of [3H]TCDD. Both HC and unmodified AC at 3% or more significantly increased fecal excretion of [3H]TCDD in comparison with the control basal diet. Consistent with this, [3H]TCDD radioactivity in the liver—a major TCDD storage organ—was markedly decreased by HC at concentrations of 3% and 10%. In an examination of potential side effects, unmodified AC at 10% or more caused significant body weight reduction and at 20% caused significant hypocholesterolemia. In contrast, HC caused weight gain reduction only at a concentration of 20%, and there was no evidence of hypocholesterolemia at any dietary HC concentration. HC not only retains the ability of AC to enhance fecal excretion of TCDD but also reduces some of the side effects of AC.
Polyhalogenated aromatic hydrocarbons (PHAHs), which include polychlorinated dibenzo-p-dioxins and dibenzofurans, are environmental contaminants found in emissions from combustion of fossil fuels; incineration of municipal, hazardous, and hospital wastes; and production of bleached paper (Swanson et al., 1988; Thoma, 1988; Clement et al., 1989; Hutzinger and Fiedler, 1989; Rosenberg et al., 1995). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototypic and most potent isomer of the PHAHs. It elicits a variety of biological and toxic responses, including induction of xenobiotic-metabolizing enzymes, endocrine disruption, behavioral abnormalities, teratogenicity, immunotoxicity, thymic atrophy, reproductive disorders, epithelial disorders, wasting syndrome, hepatotoxicity, and cancer (Goodman and Sauer, 1992; Abbott, 1995; Mimura and Fujii-Kuriyama, 2003; Alsharif and Hassoun, 2004; Carvalho and Tillitt, 2004; Miettinen et al., 2004; Puebla-Osorio et al., 2004; Simanainen et al., 2004; Uno et al., 2004; Zhang et al., 2006; Boffetta et al., 2011; Yoshida et al., 2020). TCDD tends to accumulate in the liver and adipose tissue because of its high lipophilicity (Geusau et al., 2002). In experimental animals, fecal excretion is the major route of excretion of TCDD after intratracheal instillation, gavage, or intraperitoneal or intravenous administration (Olson, 1986; Kamimura et al., 1988; Diliberto et al., 1996). However, TCDD is so hardly metabolized that it tends to remain for a long time: in humans, it has an estimated half-life of approximately 7 years (Pirkle et al., 1989; Flesch-Janys et al., 1996). Therefore, exposure to TCDD can result in serious chronic toxicity.
Humans are usually exposed to these compounds in the form of complex mixtures in the diet, particularly in milk and other dairy products and in fish and meat (Schecter et al., 1994). Some human subpopulations are at risk of either continuous or intermittent exposure to relatively high levels of PHAHs and consequently adverse health effects. For example, workers engaged in the production, use, or destruction of materials containing these chemicals or their precursors may be subject to such risks (Päpke et al., 1992; Bakoğlu et al., 2004). In the past, discrete exposures to high levels of these compounds have occurred through industrial accidents (e.g., in Seveso, Italy, in 1976), improper disposal of industrial waste (e.g., at Times Beach, Missouri, in 1982) or attempts on politicians’ lives (e.g., in the Ukraine in 2004). Additionally, the lipophilicity of dioxins and related chemicals promotes their sequestration in the adipose tissue of the breast and their concentration in breast milk during lactation (Lorber and Phillips, 2002). As a consequence, breast-fed infants can have daily exposures 10 to 20 times higher than those in the background population (Jödicke et al., 1992; McLachlan, 1993). Potential exposure of humans to TCDD has therefore aroused great concern, not only about potential toxicity, but also about how we can eliminate accumulated TCDD from the body. However, there are currently few substances available clinically for enhancing the elimination of accumulated TCDD.
Activated charcoal (AC) is used widely as antidote against most drugs and toxic poisons in cases of accidental ingestion or dosage error, because it has the potential to prevent the absorption of such toxins from the gastrointestinal tract (Levy, 1982; Yoshimura et al., 1986; McLuckie et al., 1990; Lapatto-Reiniluoto et al., 2000, 2001). Previous reports indicate that oral administration of AC increases the clearance of dioxins, including TCDD, that have already been absorbed and are in systemic circulation. In addition, TCDD absorption from the gastrointestinal tract is obstructed by AC (Manara et al., 1984; Boyd et al., 2017; Sallach et al., 2019). Although these results suggest that AC is useful for TCDD detoxification, it has some side effects, including constipation (Sato et al., 2002) and decreased serum levels of essential nutrients, including vitamin D, B12, folic acid, glucose, triglycerides, and cholesterol (Ott et al., 1979; Nakano et al., 1984a, 1984b; Kuusisto et al., 1986; Tishler et al., 1987; Park et al., 1988), because it may inhibit the absorption of these substances from the gastrointestinal tract along with dioxins. Therefore, when AC is used to reduce the absorption and accumulation of TCDD, we must be aware of the potential for deleterious effects from its long-term administration.
Alginate-coated AC, termed Health Carbon (HC), was developed as a porous material for absorbing toxins with fewer side effects of AC such as constipation. A previous report indicated that alginate stimulates colonic mucous secretion and has a tendency to relieve constipation in a rat model of spastic constipation induced by loperamide (Shimotoyodome et al., 2001). However, it remains unclear whether HC retains the ability to detoxify TCDD. Here, we demonstrate that HC enhances the fecal excretion of stored 2,3,7,8-tetrachloro [1,6-3H]] dibenzo-p-dioxin ([3H]TCDD) as effectively as AC in male mice. Consistent with this result, HC administration led to a significant reduction in [3H]TCDD-derived radioactivity in the liver. Furthermore, at the same dietary concentration at which AC caused hypocholesterolemia, HC administration did not lead to abnormal reduction of serum cholesterol levels. We discuss the potential application of HC as both an antidote for clinical use and a supplement.
[3H]TCDD was purchased from Chemsyn Science Laboratories (Lenexa, KS, USA). It had a specific activity of 33.4 Ci/mmol and a radiochemical purity of ≥ 97%. All other chemicals used were of the best commercially available grade. [3H]TCDD was diluted with olive oil. Stock solution was prepared and stored at a concentration of 250 ng/mL. Dosing solutions were prepared by dilution of stock [3H]TCDD in olive oil.
Animals and dietsEight-week-old male ddY mice purchased from SLC (Shizuoka, Japan) were used in this experiment. Mice were housed in a room maintained at 23 ± 2°C with 50% ± 10% humidity and a 12-hr light–dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). Food and water were provided ad libitum. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committees of Gifu Pharmaceutical University and Osaka University. All efforts were made to minimize both suffering and the numbers of animals used. At the time of the study, mice were housed individually in stainless-steel metabolic cages designed for the separate collection of fees and urine. AIN-93M (CLEA, Tokyo Japan) was used as the basal diet in all experiments. HC was a kind gift of Kyowa Hakko Kogyo (Tokyo, Japan), and unmodified AC was purchased from Nacalai Tesque (Kyoto, Japan).
Animal experimentsAll mice were weighed and then intraperitoneally injected on day 0 with a single dose of [3H]TCDD (0.5 µg 50 µCi–1 kg–1) dissolved in olive oil. In our examination of the toxicokinetics of [3H]TCDD in the early stages of exposure, mice were weighed on days 1, 4, 8, and 12. Food consumption were measured, and total urine and total feces were collected, days 0 to 1, 2 to 4, 5 to 8, and 9 to 12 following the single treatment. Four animals were euthanized on each of days 1, 4, 8, and 12, and blood samples were collected. Various tissues were then removed and weighed. Fecal samples were air-dried and then weighed. In our examination of the effects of HC and unmodified AC on [3H]TCDD excretion, on day 8 the mice were randomly allocated to seven groups (four mice per group). One group was fed the basal diet as a control group and the other six groups as treatment groups were given diets containing 3%, 10%, or 20% w/w of either unmodified AC or HC for 16 days. Body weight and food consumption were measured, and total urine and total feces were collected, every 2 days. Fecal samples were air-dried and then weighed. On day 24, all mice were euthanized, and blood samples were collected. Tissues were removed and weighed. All collected blood samples were immediately centrifuged and the serum in the supernatant transferred to another tube for measurement of 3H content by liquid scintillation counting, or of cholesterol levels. Samples of feces, urine, and tissues were also examined for 3H content by liquid scintillation counting.
Detection of [3H]TCDD-derived radioactivityAir-dried fecal samples were homogenized and then sampled in duplicate. Serum and fecal samples were treated with isopropanol and 30% H2O2 as a bleaching agent. All tissues and serum and fecal samples were then dissolved in Soluene-350 tissue solubilizer (Packard Bioscience, Groningen, The Netherlands). After the samples had been dissolved, Hionic-Fluor solution (Packard Bioscience) was added and radioactivity was quantified with a liquid scintillation counter. Urine samples were mixed with Clear-sol II (Nacalai Tesque) for measurement of radioactivity by liquid scintillation counter.
Detection of serum cholesterol levelSerum cholesterol levels were determined by using a Wako Cholesterol E-test kit (Wako Pure Chemical, Osaka, Japan).
StatisticsData were analyzed by using SPSS software (Chicago, IL, USA). Values with P ≤ 0.05 were considered statistically significant. Experiments were independently performed at least twice; the results were qualitatively identical, and representative experimental results are shown.
Before examining the effects of HC on [3H]TCDD excretion, we examined the toxicokinetics of [3H]TCDD in the early stages of exposure of mice. The amount of [3H]TCDD in the serum peaked on day 4. Thereafter it decreased, and it remained almost constant from day 8 onward (Fig. 1A). We then examined the daily changes in [3H]TCDD accumulation in each tissue (Fig. 1B and C). Reflecting the daily changes in its serum concentration, [3H]TCDD began to accumulate in each tissue from day 1, and the amounts accumulated became close to constant on day 4 or day 8. The liver had the greatest accumulation of [3H]TCDD as a percentage of the dose administered (Fig. 1B), whereas the accumulation per tissue weight was the greatest in adipose tissue, followed by the liver (Fig. 1C). The thymus, spleen, testis, kidney, and blood were minor sites of [3H]TCDD storage (0.02% to 0.05% of administered dose). These results suggest that the main tissues of [3H]TCDD accumulation are adipose tissue and liver, roughly in agreement with the results of previous report (Gasiewicz et al., 1983). [3H]TCDD excretion was observed in both feces and urine, but the daily percentage of the dose excreted via the feces was higher than that excreted via the urine (Fig. 1D), suggesting that TCDD was excreted mainly in the feces. Daily excretion in the feces remained almost constant from days 2 to 8 and then decreased after day 9 (Fig. 1D). In contrast, daily excretion in the urine was almost constant from day 1 (Fig. 1D). Considering these results, we determined that first-order elimination of [3H]TCDD in the early stages of exposure was terminated, and the distribution of [3H]TCDD reached a steady state, by day 8.
Toxicokinetics of [3H]TCDD following a single dose intraperitoneal administration in mice. Mice were single dose intraperitoneally injected with [3H]TCDD dissolved in olive oil and the day was set to day 0. (A) Daily profiles of serum [3H]TCDD level; (B) Daily changes in the accumulation of [3H]TCDD in each tissue as a percentage of the original dose; (C) Daily changes in the concentration of [3H]TCDD in each tissue as a percentage of the tissue weight; (D) Daily amount of [3H]TCDD excretion in feces and urine as average amount per day of each period. The results are expressed as mean ± SD (n = 4).
We then investigated the ability of HC and unmodified AC to enhance [3H]TCDD excretion. Eight days after intraperitoneal [3H]TCDD injection, a basal diet mixed with HC or unmodified AC at various percentages was fed to mice and [3H]TCDD-derived radioactivity in the feces and urine was detected. In the groups fed either HC or unmodified AC in the diet at all tested percentages, by day 24 the cumulative fecal excretion of radioactivity (average fecal excretion ranged from 17% to 22% of the administered dose) was significantly greater than that in the basal diet control group (average fecal excretion was 8% of the administered dose) (Fig. 2A and 2B). In addition, statistical analysis by Tukey’s multiple comparisons test showed no significant differences in fecal excretion of [3H]TCDD between the HC diet groups (Fig. 2A) and the unmodified AC diet groups (Fig. 2B); results of the statistical analysis are not shown. These results indicate that HC can enhance the excretion of [3H]TCDD into the feces as effectively as does unmodified AC.
Cumulative excretion of [3H]TCDD in feces and urine by [3H]TCDD-exposed mice fed with either HC or unmodified AC. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC (A and C) or unmodified AC (B and D) from day 8 to day 24. Total feces (A and B) and total urine (C and D) were collected every 2 days and then detected 3H content by liquid scintillation counting. The results are expressed as mean ± SD (n = 4) and analyzed by two-way ANOVA, followed by Dunnett’s multiple comparison test. **P < 0.01.
Across the 24-day experiment, cumulative urinary excretion of TCDD-derived radioactivity was estimated at 8% (range 7% to 13%) of the administered dose in all diet treatment groups (Fig. 2C and 2D). In the urine of guinea pigs and rats there have been similar findings of cumulative excretion of TCDD-derived radioactivity at approximately 8% (range 3% to 16%) of the administered dose (Weber et al., 1982; Olson, 1986). We found no significant differences in urinary excretion in the various groups treated with HC or unmodified AC compared with that of the basal diet. In addition, statistical analysis by Tukey’s multiple comparisons test also showed no significant differences in urinary excretion of [3H]TCDD between the HC diet groups (Fig. 2C) and the unmodified AC diet groups (Fig. 2D); results of the statistical analysis are not shown. These results suggested that neither HC nor unmodified AC had the potential to enhance TCDD excretion via the urine.
Effects of HC and unmodified AC on [3H]TCDD distribution in each tissueWe examined the distribution of [3H]TCDD in various tissues after 16 days of treatment with HC or a unmodified AC diet (Fig. 3). Compared with the basal control diet, an HC diet at 3% or 10% significantly decreased TCDD-derived radioactivity in the liver (Fig. 3A). In the group that received an HC diet at 10%, TCDD-derived radioactivity was also significantly decreased in the kidney (Fig. 3A). However, in the group that received an HC diet at 20%, there was no significant decrease in TCDD-derived radioactivity in the liver (Fig. 3A).
[3H]TCDD concentration in serum and each tissue of [3H]TCDD-exposed mice fed with either HC or unmodified AC at day 24. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC (A) or unmodified AC (B) from day 8 to day 24. At day 24, all mice were euthanized, and then serum and each tissue sample were collected. 3H content of each sample was detected by liquid scintillation counting. The results are expressed as mean ± SD (n = 4) and analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test. *P < 0.05.
Unmodified AC diet treatment at 3% or 10% tended to decrease radioactivity in the liver, but not significantly so (Fig. 3B). Thus treatment with an HC diet at suitable percentages could reduce the storage of [3H]TCDD in the liver. Notably, however, at a 20% dietary concentration of unmodified AC, but not HC, the concentration of TCDD-derived radioactivity stored in adipose tissue was strikingly and significantly increased compared with that after feeding of the control basal diet (Fig. 3). Also, at a 20% dietary concentration of unmodified AC, there was a non-significant trend toward an increase in TCDD-derived radioactivity in the liver. The opposite effect of a 20% unmodified AC diet seems to be related to extreme reduction of body weight as a side effect with the former (Fig. 4; and see the next paragraph).
Body weight gain in [3H]TCDD-exposed mice fed with either HC or unmodified AC. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC (A) or unmodified AC (B) from day 8 to day 24. Body weight of each mouse was measured every 2 days. The results are expressed as mean ± SD (n = 4) and analyzed by two-way ANOVA, followed by Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01.
To consider the potential side effects of HC, we examined its effects on weight gain, food consumption, excretion amount, and serum cholesterol level. We examined the body weight gains of mice during the 24-day experiment (Fig. 4). Significant suppression of weight gain was observed in mice given an HC diet at 20%, but not at 10% or less (Fig. 4A). In the unmodified AC treatment group, significant suppression of weight gain was observed at 10% or more, and dramatic weight loss was observed at 20% (Fig. 4B). In mice fed either HC or unmodified AC at 10%, the kidney weighed significantly more than that of the controls, despite the loss of body weight (Table 1). At 20% unmodified AC, the weights of the thymus, spleen, and liver were significantly lower than in the controls, whereas at 20% HC the weights of only the thymus were significantly lower than in the controls (Table 1).
| Tissue | Basal diet | HC | Unmodified AC | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3% | 10% | 20% | 3% | 10% | 20% | ||||||||||||||||||
| Whole body | 42.3 | ± | 4.66 | 37.9 | ± | 1.23 | 39.2 | ± | 0.92 | 35.4 | ± | 3.35a | 39.7 | ± | 1.22 | 37.8 | ± | 1.56 | 29.4 | ± | 2.61a | ||
| Thymus | 0.062 | ± | 0.008 | 0.073 | ± | 0.009 | 0.053 | ± | 0.004 | 0.047 | ± | 0.007a | 0.058 | ± | 0.005 | 0.054 | ± | 0.013 | 0.036 | ± | 0.007b | ||
| Testis | 0.25 | ± | 0.02 | 0.25 | ± | 0.01 | 0.28 | ± | 0.03 | 0.23 | ± | 0.03 | 0.25 | ± | 0.03 | 0.25 | ± | 0.01 | 0.25 | ± | 0.02 | ||
| Spleen | 0.11 | ± | 0.02 | 0.12 | ± | 0.02 | 0.13 | ± | 0.01 | 0.12 | ± | 0.03 | 0.11 | ± | 0.03 | 0.08 | ± | 0.01 | 0.07 | ± | 0.02a | ||
| Kidney | 0.60 | ± | 0.10 | 0.64 | ± | 0.03 | 0.78 | ± | 0.09a | 0.74 | ± | 0.08 | 0.69 | ± | 0.04 | 0.82 | ± | 0.10a | 0.53 | ± | 0.09 | ||
| Liver | 2.25 | ± | 0.39 | 2.00 | ± | 0.12 | 2.21 | ± | 0.09 | 2.03 | ± | 0.29 | 2.25 | ± | 0.43 | 2.19 | ± | 0.12 | 1.36 | ± | 0.23b | ||
Each value represents the mean ± 1 SD (g) of four mice per group. In the statistical analyses, one-way ANOVA followed by Dunnett’s test was conducted between the six treatment groups vs. the basal diet control. Significant differences: aP < 0.05 or bP < 0.01.
Food consumption tended to increase with both the HC treatment and the unmodified AC treatment but did not differ significantly from that in basal diet-fed mice (Fig. 5). In contrast, fecal excretion in both the HC and unmodified AC groups at 10% or more was significantly greater with dose dependency than that in the basal diet group (Fig. 6). However, there were no significant differences in total fecal dry weight between the HC and unmodified AC diet groups (Fig. 6). To determine whether the [3H]TCDD excretion enhanced by HC and AC (see Fig. 3) was simply caused by an increase in fecal excretion, we examined the concentration of [3H]TCDD in the feces. In the both HC and AC-treatment, significant increases in [3H]TCDD concentration of feces were confirmed at dosage of 3%, while there was no significant increase at dosage of 10% and more (Fig. 7). Thus the enhancement of [3H]TCDD excretion by HC and AC was not simply due to an increase in the amount of feces excreted; HC and AC were directly involved in the excretion of [3H]TCDD.
Cumulative food consumption in [3H]TCDD-exposed mice fed with either HC or unmodified AC. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC or unmodified AC from day 8 to day 24. Total food consumption in each mouse was measured every 2 days. The results are expressed as mean ± SD (n = 4) and analyzed by two-way ANOVA, followed by Dunnett’s multiple comparison test. NS; not significant.
Cumulative fecal dry weight in [3H]TCDD-exposed mice fed with either HC or unmodified AC. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC or unmodified AC from day 8 to day 24. Feces samples were collected every 2 days, and then dried and weighted. The results are expressed as mean ± SD (n = 4) and analyzed by two-way ANOVA, followed by Dunnett’s multiple comparison test. **P < 0.01.
Fecal [3H]TCDD concentration in [3H]TCDD-exposed mice fed with either HC or unmodified AC. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC or unmodified AC from day 8 to day 24. Feces samples were collected from day 9 to day 24, and then detected total 3H content for 16 days by liquid scintillation counting. The results are expressed as mean ± SD (n = 4) and analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test. **P < 0.01.
The estimated serum cholesterol level of mice in the basal diet group after the last day of the experiment (11 weeks of age, body weight 35 to 40 g) was 184.3 ± 53 mg/dL (Fig. 8). In both the HC and AC treatment groups at 10% or less, the serum cholesterol level ranged from 173 to 196 mg/dL and did not differ significantly from that in the basal diet controls. However, at 20% of the diet there was a significant, extreme reduction in the serum cholesterol level in unmodified AC-fed mice, but not in HC-fed mice, compared with the controls. Thus HC had fewer side effects in the form of reduction of body weight gain and serum cholesterol level than did unmodified AC (Fig. 8).
Serum cholesterol level in [3H]TCDD-exposed mice fed with either HC or unmodified AC at day 24. Mice intraperitoneally injected with [3H]TCDD at day 0 were given a basal diet or diets containing various concentration of either HC or unmodified AC from day 8 to day 24. At day 24, all mice were euthanized, and then each blood sample was collected for determining serum cholesterol level. The results are expressed as mean ± SD (n = 4) and analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test. *P < 0.05.
AC is a potential candidate antidote against dioxins, because an AC diet had been reported to prevent the death of mice after a single oral or intraperitoneal lethal dose of TCDD (Manara et al., 1982). In addition, an AC diet hastens the elimination of TCDD from the liver of mice (Manara et al., 1984). However, to our knowledge, the effects of AC on fecal excretion of TCDD have until now not been reported. AC inhibits the enterohepatic circulation of 2,3,4,7,8-pentachlorodibenzofuran (PenCDF) in rats, promoting the excretion of PenCDF into the feces (Kamimura et al., 1988). Our results are initial data showing that AC also enhances TCDD excretion into the feces (Figs. 2 and 3). However, a very high dose of AC led to body weight gain reduction and hypocholesterolemia (Figs. 4 and 8). One of the causes of hypocholesterolemia may be the effect of AC in reducing serum cholesterol concentrations (Friedman et al., 1978; Kuusisto et al., 1986; Tishler et al., 1987). In addition, AC decreases serum levels of essential nutrients such as fat-soluble vitamins, as well as of glucose and triglycerides (Ott et al., 1979; Nakano et al., 1984a, 1984b; Park et al., 1988); this may have contributed to the reduced body weight gain.
Until now, it was unclear whether HC, which is AC coated with alginate to decrease the constipating effect of AC, would retain the ability to detoxify TCDD, but with fewer other side effects. Our results indicated that HC, like AC, in the diet led to the elimination of TCDD-derived radioactivity via the feces at about double (P < 0.05) the rate with the basal diet. These results suggest that HC can retain the ability of uncoated AC to hasten fecal excretion of TCDD. Therefore, in HC-fed mice, it appears that TCDD reabsorption is inhibited, and TCDD excretion is promoted by the presence of the HC in the intestinal tract by a way similar to that in AC-fed mice. However, in our experiments, we did not observe a reduction in the constipating effect of AC. Although AC is reported to have a constipating effect, both AC and HC increased fecal excretion in the mice without either diarrhea or constipation, contrary to our expectation. HC and AC may both promote fecal excretion in mice, as there was no increase in the amount of food consumed by mice fed either of these diets. In addition, mice might be less likely than humans to develop constipation.
The formulation of HC are AC and alginate; however, alginate may not interrupt the adsorption ability of AC toward TCDD even if it covers in some parts of AC porous. This is considered to explain the ability of alginate to absorb TCDD; one report of an in vitro experiment indicated that alginic acid could adsorb TCDD at a high rate of 27% (Aozasa et al., 2001). It is thus probable that alginate functions as a TCDD adsorbent in places where the AC is coated by it and thus loses its adsorbent pores. In fact, our current findings indicated that HC diet could reduce the concentration of [3H]TCDD stored in the liver, even if there was an alginate coating, compared with when a basal diet was used. In addition, although a previous reports demonstrated that feeding of chow with charcoal reduces TCDD accumulation in the liver and other tissues of mice given TCDD at doses about 10 times lower than the lethal dose (Manara et al., 1982), in our experimental condition, there was not significant reduction in TCDD accumulation induced by unmodified AC diet in any tested tissues. Taken together, our observations suggest that alginate coated with AC may have a potential effect of reducing TCDD accumulation in the liver.
Nevertheless, a high AC dietary concentration of 20% significantly increased the concentration of [3H]TCDD stored in adipose tissue (Fig. 3B). This may explain some of the side effects of this high-dose treatment. A reduction in mouse body weight often induces lipid loss, which may have then resulted in increased TCDD concentration in the remaining adipose tissue. It has been reported loss of body weight and liver lipid content as a result of feeding AC for long time (12 wks) even though at not so high concentration (5%) to PenCDF-treated rats (Kamimura et al., 1988). Significant decreases in weight of the liver, thymus, and spleen in the 20% AC diet group were also observed in our experiment (Table 1).
Furthermore, our results indicated that 20% AC treatment harmed the body’s homeostasis, as seen in the extreme reduction in serum cholesterol level (Fig. 8; by 36%, P < 0.05). This reduction was likely a result of the removal of bile salts from the gut. One in vitro study has shown that bile salts are adsorbed by AC rapidly and effectively (Krasopoulos et al., 1980). Bile acids are synthesized from cholesterol through different intermediates. During the normal enterohepatic cycle, 95% to 98% of bile acids are reabsorbed and only 2% to 5% of them escape. Therefore, AC could have very effectively impaired the enterohepatic circulation of bile acid and then caused a feedback stimulation of bile acid synthesis, thus depleting hepatocyte cholesterol concentrations and reducing serum LDL cholesterol levels. AC has effectively reduced serum cholesterol and triglyceride levels in studies in hypercholesterolemic patients (Kuusisto et al., 1986; Park et al., 1988) and animals (Tishler et al., 1987). In contrast, an HC diet, even at 20%, eliminated the significant reductions of liver and spleen weight and the serum hypocholesterolemia observed with 20% doses of AC. It is probable that alginate prevents the adsorption of various essential nutrients, including bile acids and fat, to the AC under the coating.
The magnitude of the capacity of HC to detoxify TCDD in mice models suggests that this material may have a potent ability in humans to detoxify TCDD and hasten its excretion into the feces, as does AC. Long-term continuous ingestion of AC might lead to its high-level, excessive accumulation in the body. High doses of AC may be suitable for hypercholesterolemic patients but might cause hypocholesterolemia in healthy persons. Our current experiments demonstrated that not only does HC decrease TCDD accumulation in the body and stimulate TCDD excretion into the feces as well as AC, but also it has fewer side effects than AC, such as the hypocholesterolemia that may occur from high doses of uncoated AC. Therefore, in TCDD-exposed patients, it is recommended to ingest HC, instead of uncoated AC, for detoxification.
We thank Junko Hirano, Hideaki Yokoyama, Takashi Taniguchi, Jun-ichi Ishizaki, and Takuma Iguchi of Osaka University for their technical assistance with the experiments in the early stages of this work. We also thank KYOWA HAKKO KOGYO Co., Ltd. (Tokyo, Japan) for providing the Health Carbon. This work was supported in part by a research Grant from the Ministry of Health, Labor, and Welfare, Japan (21KD1004) and The Japan Food Chemical Research Foundation.
Conflict of interestThe authors declare that there is no conflict of interest.
