Breast milk carotenoids provide neonates with a source of vitamin A and potentially, oxidative stress protection and other health benefits. Chlorella, which has high levels of carotenoids such as lutein, zeaxanthin and β-carotene, is an effective dietary source of carotenoids for humans. In this study, the effect of maternal supplementation with Chlorella on carotenoid levels in breast milk at early lactation was investigated. Ten healthy, pregnant women received 6 g of Chlorella daily from gestational week 16-20 until the day of delivery (Chlorella group); ten others did not (control group). Among the carotenoids detected in breast milk, lutein, zeaxanthin and β-carotene concentrations in the Chlorella group were 2.6-fold (p = 0.001), 2.7-fold (p = 0.001) and 1.7-fold (p = 0.049) higher, respectively, than those in the control group. Our study shows that Chlorella intake during pregnancy is effective in improving the carotenoid status of breast milk at early lactation.

Chlorella (Parachlorella beijerinckii) on the excretion and tissue accumulation of methylmercury (MeHg), we orally administered 5 mg/kg of MeHg chloride (4 mg Hg/kg) to female C57BL/6N mice (aged 10 weeks). The mice were housed in metabolism cages to collect urine and feces for 3 weeks with diets containing 0%, 5%, or 10% P. beijerinckii powder (BP) in -increases in the cumulative Hg eliminations into urine (5% BP) and feces (5% and 10% BP) were found in the BP groups. Twenty-one days after administration, the organ Hg levels in both BP groups tended to decrease compared to that of the control group. The reduction of Hg levels in the kidney and brain were to glutathione (GSH) metabolism, no difference was found in GSH levels in the blood or organs between the control group and the 10% BP group. These results suggest that continuous BP intake accelerates the excretion of MeHg and subsequently decreases tissue Hg levels in mice, with no alteration of GSH metabolism. We should conduct further research to elucidate details regarding the mechanism of BP-induced enhancement of MeHg excretion.

-Chlorella (Parachlorella beyerinckiiChlorella vulgaris CK-5, is a unicellular green algae that has for many years been used as a nutritional supplement. In order Chlorella, we examined the absorption and excretion of MeHg in mice. Female C57BL/6N mice were randomly divided into three groups 5 mg (4 mg Hg)/kg body weight with or without 100 mg/mouse of P. beyerinckii powder (BP), and were assigned to either a MeHg group or MeHg + BP group, accordingly. Twenty-four hr after oral administration, feces and urine were collected, and blood, liver, and kidney samples were obtained. Total mercury contents in the samples obtained were determined using an atomic absorption method. The amounts of Hg excreted in feces and urine of the MeHg + BP group were increased nearly 1.9 and 2.2-fold compared -ent between two groups. These results suggest that the intake of BP may induce the excretion of Hg both in feces and urine, although it does not affect MeHg absorption from the gastrointestinal tract. The effect of BP on the tissue mercury accumulation may become evident in a long-term experiment.Parachlorella beyerinckii, MeHg, Chlorella

The effect of Parachlorella beyerinckii CK-5, previously identified as Chlorella vulgaris, on gastrointestinal absorption of lead was investigated in mice. Female ICR mice aged 7 weeks were orally administered lead acetate solution at doses of 20 mg and 40 mg of lead per mouse, with or without 100 mg of P. beyerinckii powder (BP). The mice were bred for 24 hours. The amount of lead excreted in feces within 24 hours, and the lead levels of the blood, liver and kidney were analyzed by atomic absorption spectrometry. The percentage of total fecal excretion in mice administered BP increased by 27.7% in 20 mg lead administered mice and 17.2% in 40 mg lead administered mice in comparison to control mice, respectively. On the other hand, the lead levels of the blood, liver and kidney of BPadministered mice at 24 hours after lead administration were 48-63% lower as compared with those of control mice. The lead adsorption ability of BP and the pepsin non-digestive residue of BP (dBP) were investigated in vitro. One hundred mg of BP and dBP could adsorb 10.6 mg and 6.0 mg of lead in a 20 mg per 10 mL of lead solution, respectively. The lead absorption abilities of BP and dBP were considered to contribute to the prevention of gastrointestinal absorption of lead and the promotion of the excretion of lead. These results suggested that BP treatment might be useful in animals and humans exposed to lead.

-Methylmercury (MeHg) is gradually changed to inorganic Hg after demethylation in animal tissues, and a selective quantification of inorganic Hg in the tissues is necessary to detect the reaction. We detected inorganic Hg formation in liver and kidney of mouse as early as 24 hr after MeHg injection. As an example of biological demethylation, the cytochrome P450 (P450)-mediated N-demethylation of drugs has been well documented, and formaldehyde was detected as a reaction product. Here we incubated mouse liver homogenate with added MeHg and observed a dose-dependent production of formaldehyde, as well as inorganic Hg formation. Since the amount of formaldehyde was approx. 500 times higher than that of the inorganic Hg that formed, the formaldehyde production would be stimulated by inorganic Hg formed from MeHg. We observed that inorganic Hg caused formaldehyde production, and it was enhanced by L-methionine and sarcosine. Thus, some biomolecules with S-methyl and N-methyl groups may function as methyl donors in the reaction. Using subcellular fractions of mouse liver, we observed that microsomal P450 did not participate in the demethylation of MeHg, but the greatest activity was located in the mitochondria-rich fraction. The addition of superoxide anion in the reaction mixture significantly enhanced the formaldehyde production, whereas Mn-superoxide dismutase depressed the reaction. Our present findings demonstrated that inorganic Hg formed by MeHg demethylation in mouse liver stimulated the endogenous formaldehyde production, and we observed that MeHg demethylation could be estimated by a formaldehyde analysis. Our results also suggested that superoxide anion is involved in the reaction.

-An open-label clinical trial was performed to test the effects of unicellular green alga Chlorella supplementation on mercury concentrations of hair and blood in healthy subjects. Fifty-eight healthy participants (36 male and 22 female) were assigned to Chlorella and control groups. The Chlorella group of 35 subjects received Chlorella tablets (9 g/day) for an experimental period of 3 months while the control group of 23 subjects did not. Total mercury concentrations of hair and blood were analyzed at the beginning and end of the experimental period for estimation of methylmercury (MeHg) levels in the body. The hair mercury concentration of the Chlorella group (n = 33) was significantly decreased during the experimental period (p = 0.041) while the change in the control group (n = 23) was not significant (p = 0.362). Although the decrease in blood mercury concentration in the Chlorella group (n = 19) was not significant (p = 0.084), the change of values (values at end -values at beginning) in this group was significantly greater than that in the control group (n = 20, p = 0.038). The fish intake rates remained relatively constant during the experimental period in both the Chlorella and control groups. These results suggest that supplementation with Chlorella for 3 months in healthy subjects might reduce their body MeHg levels.

The aim of this study was to evaluate the safety of Chlorella vulgaris CK-22 as a food supplement. We examined mutagenicity, acute toxicity and subacute toxicity using Wistar rats administered Chlorella powder (CP). In the mutagenesis test, CP exhibited no mutagenecity in the in vitro assay. In the acute toxicity test, CP was administered orally at 0 mg/kg, 1,000 mg/kg, 2,000 mg/kg and 5,000 mg/kg body weight to Wistar rats (five animals/sex/group). No significance changes were observed test article-related during the 14-day observation period. The LD 50 of CP was estimated to be more than 5,000 mg/kg body weight in rats. In the subacute toxicity test, CP was administered at 0%, 2.5%, 5% and 10% in pelleted rodent diet to Wistar rats (ten animals/sex/groups). No mortality or treatment-related clinical signs were observed in any of the groups during the 28-day observation period. In both sexes, renal histopathology was conducted in the control and 10% groups, because absolute and relative renal weights increased in the 10% groups compared to the control groups. Based on the histopathology of the kidneys, the no-observed-adverse-effect level (NOAEL) is greater 8.57 g/kg body weight/day for males and 8.62 g/kg body weight/day for females.

The genus Chlorella contains unicellular green algae that have been used as food supplements. The purpose of this work was to evaluate the safety of the Chlorella sorokiniana strain CK-22 using powdered preparation (CK-22P) both by in vitro and in vivo assays. These included an experiment for cytotoxicity using Chinese hamster lung fibroblasts (V79 cells) and a 13-week repeated-dose oral toxicity trial using Wistar rats. The cytotoxicity was evaluated by MTT assay of a hot water extract (Hw-Ex) and 80% ethanol extract (Et-Ex) of CK-22P, and no effect on cell viability was observed. The 50% viability inhibitory effect (IC50) value for Hw-Ex and Et-Ex were estimated as greater than 73 and 17 μg/ml, respectively. In the subchronic toxicity test, pelleted rodent diet containing 0%, 2.5%, 5% or 10% CK-22P was given to Wistar rats (ten animals/sex/groups) for 13 weeks. During the experimental period, no CK-22P treatment-induced differences in general condition, body weight gain, food and water consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, histopathology, or animal death were observed. The no-observed-adverse-effect levels (NOAEL) were estimated to be 5.94 g/kg body-weight/day for males and 6.41 g/kg body-weight/day for females.