Distinct roles of HMGB1 in the regulation of P­glycoprotein expression in the liver and kidney of mice with lipopolysaccharide­induced inflammation.

The role of high mobility group box 1 (HMGB1) in the regulation of efflux transporters in the liver and kidney remains unclear, although it has been reported that HMGB1 can increase P­glycoprotein (P­gp) expression in the brain. The present study aimed to clarify the involvement of HMGB1 in the regulation of P­gp expression in the liver and kidney of mice with lipopolysaccharide (LPS)­induced inflammation. Mice were treated with LPS or LPS + glycyrrhizin (GL); GL is as an HMGB1 inhibitor. Subsequently, the expression levels of transporters, such as P­gp, and HMGB1 receptors, such as toll­like receptor (TLR)4 and receptor for advanced glycation end­products (RAGE), were determined by quantitative PCR and LC­MS/MS­based targeted proteomics. For the in vitro study, HepG2 and KMRC­1 cells were used, as was a co­culture of KMRC­1 and differentiated THP­1 cells. The mRNA and protein expression levels of Mdr1a and Tlr4 in the kidneys of LPS + GL­treated mice were significantly decreased compared with those in LPS mice. The results indicated that HMGB1 had little effect on the expression of Mdr1a and Tlr4 in the liver, since there was little change in of Mdr1a and Mdr1b expression between the LPS and LPS + GL­treated mice. Notably, regarding MDR1 mRNA expression, KMRC­1 cells were more responsive to LPS than HepG2 cells, and KMRC­1 cells treated with LPS exhibited increased levels compared with control KMRC­1 cells. In differentiated THP­1 cells, LPS treatment decreased the mRNA expression levels of TLR4, whereas they were restored to control levels by HMGB1. In conclusion, HMGB1 in the plasma and TLR4 in macrophages may be involved in the regulation of P­gp expression in the kidneys of inflamed mice.

Failure to gulp surface air induces swim bladder adenomas in Japanese medaka (Oryzias latipes).

In order to elucidate the effects of swim bladder inflation failure on swim bladder carcinogenesis, we investigated the sequential histopathological changes of swim bladders at 13, 24, 35, and 53 days post-hatch (dph) in medakas with an uninflated swim bladder, which was experimentally induced by denying access to the air-water interface between 0 and 6 dph. The reactive oxygen species (ROS) levels were measured at 24 dph. An uninflated swim bladder was induced in 47.3% of the fish denied access to the air-water interface (the denied group). The total incidence of swim bladder adenoma was 54.1% in the denied group; however, these tumors were observed in all fish with an uninflated swim bladder. In fact, these tumors were observed from 13 dph and onwards. The TBARS levels of the juveniles showed a 2.6-fold increase in fish with an uninflated swim bladder in the denied group compared to that in the control group. It is speculated that swim bladder inflation failure has some effects on the gas gland to produce ROS, leading to DNA damage in the gas glandular epithelium, which develops into swim bladder adenomas. Consequently, it is concluded that denying access to the air-water interface between 0 and 6 dph in medaka is an easy method of inducing swim bladder tumors in a short-term period, and is a useful method for producing tumor-bearing fish.

Spontaneous seminoma in medaka (Oryzias latipes).

Although spontaneous development of seminoma is rare in medaka, we encountered spontaneous testicular tumors located within the abdominal cavity in two adult medakas. The growth patterns of the tumors were a combination of solid and cord arrangements in one of the two cases (Case I) and lobular in the other case (Case II). The tumor cells resembled the cells at different stages of spermatogenesis, and a small number of oocyte-like cells were also scattered within the tumor. The tumor with solid and cord patterns showed loss of normal testicular architecture, and the tumor cells had partly invaded the dorsal muscular tissue and metastasized to the liver, kidney, and eye. The tumor with a lobular pattern did not exhibit local invasion or metastasis. The tumors were diagnosed as seminomas based on their histopathological characteristics, and the tumor in Case I was observed to be more malignant than that in Case II.

Increased penetration of diphenhydramine in brain via proton-coupled organic cation antiporter in rats with lipopolysaccharide-induced inflammation.

Uptake transporters in brain microvascular endothelial cells (BMECs) are involved in the penetration of basic (cationic) drugs such as diphenhydramine (DPHM) into the brain. Lipopolysaccharide (LPS)-induced inflammation alters the expression levels and activities of uptake transporters, which change the penetration of DPHM into the brain. A brain microdialysis study showed that the unbound brain-to-plasma partition coefficient (K p,uu,brain) for DPHM in LPS rats was approximately two times higher than that in control rats. The transcellular transport of DPHM to BMECs was increased when BMECs were cultured with serum from LPS rats. Compared with control rats or BMECs, the brain uptake of DPHM in LPS rats was increased and the intracellular accumulation of DPHM was increased under a high intracellular pH in BMECs from LPS rats, respectively. Treatment of BMECs with transporter inhibitors or inflammatory cytokines had little impact on the intracellular accumulation of DPHM in BMECs. This study suggests that LPS-induced inflammation promotes unidentified proton-coupled organic cation (H+/OC) antiporters that improve the penetration of DPHM into rat brain via the blood-brain barrier.

Swim bladder tumor in the young adult scoliotic medaka (Oryzias latipes).

A swim bladder tumor was detected in one scoliotic medaka aged 22 weeks. The tumor was located in the dorsal abdominal cavity, with maximum dimension of 1,850 × 1,500 µm. No swim bladder lumen was identified, and the region where the swim bladder lumen would have been located, was replaced with adipose tissues. The tumor was a non-invasive, expansile, and encapsulated solid mass with a few cysts, and comprised a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor mass was connected to a rete mirabile that showed a hyperplastic capillary plexus; however, the tumor cells did not invade the rete mirabile, thereby revealing that the tumor was an adenoma originating from the gas glandular epithelium of the swim bladder. Since proliferative lesions in the swim bladder have been reported in some teleosts with skeletal deformations, including medaka, the occurrence of a spontaneous swim bladder tumor in teleosts is considered to be closely associated with various types of skeletal deformation, and spinal curvature in particular.

Swim bladder tumors in the wavy medaka (Oryzias latipes).

Swim bladder tumors were detected in three out of 28 wavy medakas aged about 2 years old, all of which displayed abnormal swimming patterns caused by their spinal curvature. The tumors were located in the dorsal abdominal cavity. The swim bladder lumen was not detected in the region where it was originally assumed to be located, and that region was replaced with adipose tissue. The tumors were non-invasive, expansile, and encapsulated solid masses composed of a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor masses were connected to the rete mirabile, but the tumor cells did not infiltrate into them. Histopathologically, these tumors were diagnosed as adenomas originating from the gas glandular epithelium of the swim bladder. Spontaneous swim bladder tumors are rare in medaka, with an incidence of 0.02%; however, in the present study of wavy medaka, the incidence was much higher (10.7%). The long-term physical effects on the gas gland caused by swim bladder deformation considered to be a secondary effect of the spinal curvature may be an important factor in the proliferation of the gas glandular epithelium in the wavy medaka, resulting in the higher incidence of swim bladder tumors.

Sequential histological changes in the liver of medaka exposed to methylazoxymethaol acetate.

We performed a medaka bioassay for the carcinogenicity of methylazoxymethaol acetate (MAM-Ac) to examine the sequential histological changes in the liver from 3 days after exposure until tumor development. The medaka were exposed to MAM-Ac at a concentration of 2 ppm for 24 hours, and were necropsied at 3, 7, 10, 14, 21, 28, 35, 42, 49, 60, and 91 days after exposure. MAM-Ac induced four cases of hepatocellular adenoma and one case of hepatocellular carcinoma in 8 fish after 60 or 91 days of exposure. Histological changes in the liver until tumor development were divided into three phases. In the cytotoxic phase (1-10 days), MAM-Ac-exposed hepatocytes showed vacuolar degeneration and underwent necrosis and apoptosis, resulting in multiple foci of hepatocyte loss. In the repopulation phase (14-35 days), the areas of hepatocyte loss were filled with hepatic cysts and the remaining hepatocytes were surrounded by hepatic stellate-like cells (or spindle cells) and gradually disappeared. In the proliferation phase (42-91 days), the original hepatic parenchyma was regenerated and progressively replaced by regenerative hyperplastic nodules and/or liver neoplasms. The medaka retained a strong hepatocyte regenerative ability in response to liver injury. It is considered that this ability promotes the proliferation of initiated hepatocytes in multistep carcinogenesis and influences the development of liver tumor over a short period in medaka.

Histopathology of a wavy medaka.

Wavy medakas are medakas that exhibit spinal curvature characterized by dorsoventrally curved vertebrae. We found a spontaneous wavy medaka in our experimental stock and subjected it to a histopathological examination. Macroscopically, the wavy medaka's spine formed an M shape, and its vertebrae displayed a dorsoventral curvature that started at the third vertebral bone. Microscopically, the vertebral cavities were filled with fibrous tissue, which was similar to that seen in the central parts of the intervertebral discs of a normal medaka. The vertebral joints were composed of vacuolated notochord cells without intervertebral disc formation. These changes were also observed in the caudal region, which exhibited less curvature. In the normal medaka, the intervertebral discs form via the regression of the notochord that plays a key role in the development of vertebrae and disc formation. We concluded that notochordal subinvolution had induced intervertebral disc dysplasia, leading to lordokyphosis, in the wavy medaka.

Effect of chlorpromazine on rat placenta development.

We examined the sequential histopathological changes in the placentas from rats exposed to chlorpromazine. Chlorpromazine was intraperitoneally administered on GD 14 at 50 and 100 mg/kg and the placentas were sampled on GDs 14.5, 15, 17 and 21. The incidence of dams with complete fetal resorption was increased from GD 17 up to 20% at 50 mg/kg and 44.4% at 100 mg/kg. The embryo/fetal weights reduced on GDs 15 and 17 at 50 mg/kg and during GDs 15-21 at 100 mg/kg. The placental weights reduced on GD 17 at 50 mg/kg and during GDs 14.5-21 at 100 mg/kg. Histopathologically, in the labyrinth zone, apoptotic cells were scattered in the trophoblastic septa without inhibition of cell proliferation on GDs 14.5 and 15 at 50 and 100 mg/kg in a dose-dependent manner. A decrease in trophoblasts led to labyrinth zone hypoplasia. In the basal zone, apoptotic cells were scattered on GDs 14.5 and 15 at 100 mg/kg, and most of them appeared to be glycogen cells. A decrease in glycogen cells induced the delayed development of glycogen cell islands and the subsequent remaining glycogen cell islands, and led to the cystic degeneration of glycogen cells. In addition, failure of development of the glycogen cell islands led to the impaired interstitial invasion of the glycogen cells, and then metrial gland hypoplasia occurred.

Background data on developmental parameters during the gestation period in rats.

Background data during the gestation period were obtained from 128 Wistar Hannover GALAS rats and 26 Crl:CD(SD) pregnant rats in the control groups of our previous toxicity studies. The body weights of dams in the Wistar Hannover GALAS rats were significantly lower throughout the gestation period than those in the Crl:CD(SD) rats. In contrast, the time-dependent change in the body weight gain (%) of dams showed very similar trends in both strains. The mean number of live embryos/fetuses in the Wistar Hannover GALAS rats was 12.0, and was lower than that (14.5) in the Crl:CD(SD) rats. The placental weights gradually increased with pregnancy progression and reached a plateau on gestation day (GD) 19, although the embryo/fetal weights rapidly increased from GD 17 to GD 21. The embryo/fetal weights in the Wistar Hannover GALAS rats were significantly lower on only GD 21 than those in the Crl:CD(SD) rats. It is considered that this fetal weight difference between the strains develops during the fetal period, but not during the organogenesis period. In contrast, there were no differences in the placental weights between the two strains. Microscopically, the thickness of the labyrinth zone in the Wistar Hannover GALAS rats was thicker throughout the gestation period than that in the Crl:CD(SD) rats.