Various cytotoxic drugs were purchased, as follows: carbon tetrachloride (CCl4), salicylic acid, ferric sulfate, sodium valproate, N-nitroso-fenfluramine, and D-galactosamine were purchased from Wako Pure Chemical Industries, Osaka, Japan; acetaminophen was purchased from Tocris Biosciences, Ellisville, MO; acetylsalicylic acid was purchased from Cayman Chemical Co., Ann Arbor, MI; tetracycline was purchased from LKT Laboratories Inc., St Paul, MN; amiodarone hydrochloride was purchased from MP Biomedicals, Solon, OH; methyldopa was purchased from Sawai Pharmaceutical Co., Ltd., Osaka, Japan; ketoconazole was purchased from LKT Laboratories Inc.; and pemoline was purchased from Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan. N-acetyl-L-cysteine (NAC), which acts to suppress increases in ALT activity, was purchased from Sigma-Aldrich, St. Louis, MO. Hydrosoluble and liposoluble compounds were dissolved in saline and dimethyl sulfoxide, respectively.
Fertilized silkworm eggs (Bombyx mori, Hu·Yo × Tukuba·Ne) were purchased from Ehime Sanshu Co., Ltd. (Ehime, Japan). Hatched larvae were fed artificial food, Silkmate 2S (Nosan Corporation, Yokohama, Japan) at 27°C.
Construction of cytotoxic induction model using silkworm larvae
Fifth-instar silkworm larvae on the first day were fed artificial food, Silkmate 2S, for 1 d. After the body weight increased to 1.8 to 2.2 g, they were fasted for 6 h, and solution containing a cytotoxic compound was injected into the hemocoel from the backside of the larvae. Liposoluble compounds were injected (25 μL/silkworm) using a glass syringe (MICROLITERTM #710, Hamilton Co., Reno, NV) with a 27G needle, and hydrosoluble compounds were injected (50 μL/silkworm) using a disposable syringe (Terumo Corporation, Tokyo, Japan) with a 27G needle. After incubation at 27°C for 1 d, the hemolymph was collected for measurement of ALT activity as described below.
Examination of suppressive effects against induced cytotoxicity
Fifth-instar silkworm larvae on the first day were fed Silkmate 2S for 1 d. After the body weight increased to 1.8 to 2.2 g, they were fasted for 6 h, and 50 μL of 0.9% saline or 0.4 M NAC was injected into hemocoel from the backside of the larvae using a disposable syringe. After 30 min, 25 μL of olive oil or 15% CCl4 was injected into the hemocoel using a glass syringe. After incubation at 27°C for 1 d, the hemolymph was collected for measurement of ALT activity as described below.
Preparation of tissue homogenates from silkworm larvae
Fifth-instar silkworm larvae on the first day were fed Silkmate 2S for 1 d. After fasting for 6 h, the gut, fat body, silk gland, Malpighian tube, and outer coat were isolated. Each tissue was weighed and homogenized with insect physiologic saline (150 mM NaCl, 5 mM KCl, 1 mM CaCl2). Samples were centrifuged at 3000 rpm for 5 min, and the supernatant was collected and stored at −80°C until measurement of ALT activity. The amount of protein in the supernatant was quantified using Lowry’s method.
Measurement of ALT activity
Five μL of collected hemolymph or the supernatant of homogenized tissue was mixed with 550 μL of a reaction solution containing 0.5 M L-alanine, 0.2 mM NADH, 1.3 U/mL lactate dehydrogenase, and 0.9 mg/mL bovine serum albumin. After adding 50 μL of 180 mM 2-oxoglutarate solution, the reaction mixtures were incubated at 30°C for 90 min. Absorbance at 339 nm was recorded to detect decreases in NADH. The slope of the absorbance decrease is proportional to ALT activity. Final ALT activities were determined according to the standard curve drawn from the results of mouse liver homogenate. For ALT activity, 1U was defined as the enzyme activity that forms 1 μmol NAD/min under the assay conditions.
All experiments were performed at least twice and the data are shown as the mean ± standard deviation. The significance of differences was calculated using a 2-tailed Student's t-test at the significance level alpha = 0.05.