Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation.

The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-I) to metaphase II (M-II) and M-II at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-I to M-II stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

Amino acids in maturation medium and presence of cumulus cells at fertilization promote male pronuclear formation in porcine oocytes matured and penetrated in vitro.

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.

[Traditional remedy-induced Chinese herbs nephropathy showing rapid deterioration of renal function].

A 19-year-old female was referred to our hospital for azotemia and anemia. She had been taking a health food for atopic dermatitis for about three years. Urinalysis showed proteinuria, glycosuria and microscopic hematuria. Generalized aminoaciduria was observed. Moreover, severe anemia, azotemia, hypokalemia and hypophosphatemia were also observed. Renal biopsy specimen disclosed hypocellular interstitial fibrosis and degeneration of the proximal tubular epithelial cells. No remarkable changes were observed in the glomeruli. Aristolochic acid was detected in the health food. From these findings, she was diagnosed as having Chinese herbs nephropathy (CHN). Although consumption of the food intake was stopped, her renal function deteriorated rapidly. Previously, we reported that certain kinds of Chinese herbal drugs contain aristolochic acid and that the drugs should be prohibited if aristolochic acid is identified. However, we experienced a patient of CHN arising from traditional remedy, which was not proved to be safe. It should be awared that health foods may contain aristolochic acid.

Prognostic evaluation of preoperative combined treatment for advanced cancer in the lower rectum with radiation, intraluminal hyperthermia, and 5-fluorouracil suppository.

BACKGROUND: Since 1977, we have studied preoperative treatments using 5-fluorouracil (5-FU) suppositories alone or in combination with radiation to reduce local recurrence and improve survival of patients with rectal cancer. PATIENTS AND METHODS: We developed a novel preoperative therapy combining radiation, intraluminal hyperthermia, and 5-FU suppositories. Thirty-five patients with rectal cancer underwent surgery after this treatment, and 41 patients underwent surgery without pretreatment. RESULTS: The patients who underwent preoperative combination treatment showed significant tumor reduction; 2 achieved microscopically complete regression. In comparison with the patients who underwent surgery without pretreatment, the preoperative treatment group had a 16.7% lower local recurrence rate (10.4% versus 27.1%) and an improved survival rate (81.8% versus 67.6%). CONCLUSIONS: Preoperative combination therapy is a promising modality for the patients with respectable advanced cancer in the lower rectum.

[Clinico-pathological studies on three preoperative combined treatments for rectal cancer].

To prevent postoperative local recurrence of rectal cancer, we treated patients using preoperative hyperthermia (5-6 times), irradiation (total 30 Gy) and 5-fluorouracil suppository (2,000-2,500 mg). The subjects were 31 patients given combined treatments and 28 patients given surgery alone. The results were as follows: 1. Histologically, therapeutic effects were recognized in 80.6% of patients receiving combined treatments. 2. The mean distance from the adventitia to the site of cancer infiltration was 6.54 mm in the combined treatments group and 3.35 mm in the surgery alone group. The difference between the two was significant (p < 0.05). 3. The rate of local recurrence in the combined treatments group was less than that in the surgery alone group. 4. No systemic side effects nor severe complications were observed during hospitalization in the combined treatments group. 5. The survival rate of the combined treatments group was higher than that of the surgery alone group. It was considered that combined preoperative treatments for rectal cancer were beneficial to survival and local control.

Intraarterial infusion chemotherapy for metastatic liver tumors using multiple anti-cancer agents suspended in a lipid contrast medium.

An ethiodized oil, Lipiodol (Lipiodol Ultra-Fluid, Laboratoire Guerbet, Paris, France), when injected into the hepatic artery, is selectively retained by liver tumors. Thus, anti-cancer agents suspended in Lipiodol can be delivered specifically to liver tumors. Before clinical trials of this new drug delivery system were begun, the movements of drugs suspended in Lipiodol were examined in vitro. It was found that 5-fluorouracil, doxorubicin, and mitomycin C were continuously released from oil phase to water phase, and when these three drugs were collectively suspended in Lipiodol, each was released independently. During clinical investigations, 42 patients with unresectable metastatic liver tumors were treated with intraarterial infusions of anti-cancer drugs in Lipiodol. As a result, the administration of a combination of anti-cancer agents suspended in Lipiodol was shown to be effective in controlling metastatic liver tumors. It was concluded that such a method could be applied to a variety of metastatic liver tumors with different drug sensitivities.

[Clinical study of the drug delivery in metastatic liver cancer using bromodeoxyuridine].

As the model of an anti-cancer agent acting at DNA synthesizing phase, BrdU was infused for metastatic liver cancers into the portal vein and/or the hepatic artery. After administration, the liver tumors were resected, and immunohistochemical staining was performed using anti-BrdU monoclonal antibody. Two of 6 portal group tumors were stained. All 4 arterial and 3 arterio-portal group tumors were stained. Therefore, it was proved that metastatic liver tumors had both arterial and portal blood supply. But areas to which BrdU was delivered were only peripheral, and the inmost area was 2 mm from the tumor surface. After the intra-arterial infusion of BrdU suspended in lipiodol, the delivery of BrdU was highly enhanced and 4 of 6 tumors were stained even to the deepest areas. However, one tumor of which the major part was necrotic and one tumor which produced mutin were not stained at all. It was interesting that after intra-arterial administration of BrdU suspended in lipiodol, BrdU was also found in the cytoplasm of normal liver cells.

[Cytofluorometric and histopathological studies on non-cancerous liver lesions after lipiodol-transcatheter arterial embolization].

We have investigated the effects of preoperative Lipiodol-Transcatheter arterial embolization (Lp-TAE) on both the nuclear DNA content of noncancerous liver cells by DNA-cytofluorometry (using NIKON SPM-RF1-D), and the histopathological findings of liver lesions. Lp-TAE through the hepatic artery was performed on ten primary hepatocellular carcinoma patients using Gelfoam in combination with adriamycin (40 mg) or mitomycin C (20 mg), emulsified in Lipiodol (10 ml) and 60% Urografin (2 ml). And hepatic resection was carried out after one or two months later in each case. Histologically, primary hepatocellular carcinoma lesions showed various patterns of necrosis, while the non-cancerous liver cells did not show hepatocellular infarction in the liver cirrhosis. The results of the nuclear DNA content showed an increase in the fractions of poliploid cells in the non-cancerous lesions treated with Lp-TAE compared with that in the normal age-matched controls. But we found no remarkable differences in the ploidy patterns between Lp-TAE treated patients and untreated patients with liver cirrhosis. In conclusion, preoperative Lp-TAE does not induce any remarkable histological hepatocellular damage of non-cancerous lesion and does not affect nuclear DNA content of non-cancerous liver cells.

[Trans-arterial embolization or transarterial infusion of anti-cancer drugs suspended in a lipid contrast medium against unresectable metastatic liver cancer].

We had 84 patients with unresectable metastatic liver cancer from May, 1980 to December 1987 in the National Matsudo Hospital. Eighteen of them had no treatment; 26 of them had serial transarterial embolization (TAE) with or without infusion of anti-cancer drug dispersed in lipiodol; 7 of them had only serial trans-arterial infusion (L-TAI) of an anticancer drug suspended in lipiodol. A significant survival advantage of patients with TAE or L-TAI was noted when compared with no treatment, but there was no difference between the survival rate of patients with TAE and L-TAI. There were some 82 patients with unresectable metastatic liver cancer from October 1984 to March 1988 in Kyto Prefectural University of Medicine Hospital. Thirteen of them had L-TAI using one anti-cancer drug (one-drug group); 8 of them had L-TAI using two drugs (two-drug group); 22 of them had L-TAI with three drugs (three-drug group). The survival rate of the three-drug group was superior to the one- and two-drug groups. It was concluded that TAE was not necessary for metastatic liver cancer, but L-TAI must be undertaken, because combined cancer chemotherapy enhanced the effect of L-TAI.

[Microscopic changes in the human liver after intra-arterial infusion of anticancer agents suspended in lipiodol].

Microscopic injury to the human liver after an intraarterial infusion of anti-cancer agents suspended in lipiodol has been evidenced by Hematoxylin-Eosin staining and by Oil Red O staining. In cases involving primary hepatic tumors, the liver tumor tissue has been injured severely while normal tissue showed no injury. In cases involving metastatic liver tumors, the tumor tissue, and also the normal liver tissue was injured though the latter injury was only mild. However, when multi-anticancer agents that were suspended in lipiodol were infused intra-arterially, the normal liver tissue was injured, even from a single drug suspension.

[Neo-adjuvant therapy in childhood osteogenic sarcoma: a pilot study of selective postoperative chemotherapy based on response to preoperative high-dose methotrexate].

"Neo-adjuvant therapy" with preoperative high-dose methotrexate (HD-MTX) and CF rescue therapy was investigated in four children with osteogenic sarcoma. Immediately after the diagnosis of osteogenic sarcoma from biopsy, the patients were treated with three to five courses of weekly HD-MTX (300 mg/kg) with CF rescue. Three patients had en bloc tumor resection and one patient underwent disarticulation of the hip joint after the pre-operative HD-MTX. The effect of HD-MTX was evaluated on the basis of pathological changes between the specimen of the primary tumor taken at biopsy and that during surgery. Two out of four patients showed marked tumor cell reduction (greater than 50%) of the specimen upon surgery. Two patients who responded to the preoperative HD-MTX were further treated with HD-MTX on a post-operative adjuvant therapy basis for 18 months. Both of these patients survived with no evidence of disease for 35.6+ and 20.9+ months. Two patients who responded poorly to HD-MTX were treated with a multi-drug postoperative adjuvant therapy including cis-platinum, adriamycin, cyclophosphamide, actinomycin D, and bleomycin. One patient had a solitary lung metastasis at 12.2 months after amputation. Wedge resection of the metastatic tumor was performed and adjuvant therapy with cis-platimum has been given for 20 months. He has remained with no evidence of disease for more than 30 months. Another patient has been receiving multi-drug neo-adjuvant therapy without any evidence of disease for 11.9 months after surgery. These data suggest that neo-adjuvant chemotherapy based on the response to preoperative HD-MTX is more useful for increasing the cure rate of childhood osteogenic sarcoma.