Experience of freezing human oocytes using sodium-depleted media.

Human embryo cryopreservation techniques enable the storage of surplus embryos created during assisted reproduction procedures; however, the existence of these same surplus embryos has sparked further debate. What can be their fate once they are no longer desired by their parents or if the parents are deceased? Thus, the level of interest in the cryopreservation of oocytes has increased, as has the necessity for further scientific study. This study had the objective of reporting 10 years of experience of freezing and thawing human oocytes from patients who did not wish to freeze embryos. A total of 159 cycles using frozen­thawed oocytes were performed (mean age 33.7 years). Survival and fertilization rates were 57.4% and 67.2%, respectively. Cleavage rate was 88.4% and the pregnancy rate was 37.7%. Clinical pregnancy was observed in 43 cycles (27.0%) with 14.5% of transferred embryos implanted. These pregnancies delivered 19 boys and 23 girls, two pregnancies are ongoing and nine were miscarriages. The average gestational week was 37.6 weeks and birthweight was 2829.2 g. These data suggest that the use of frozen­thawed oocytes in IVF represents a reasonable alternative for those patients not comfortable with the cryopreservation of supernumerary embryos.

Successful birth after injection of frozen human oocytes with frozen epididymal spermatozoa.

A couple (female 31, male 42 years old) with infertility due to obstructive azoospermy returned to the clinic in order to attempt pregnancy using their frozen oocytes and epididymal sperm cells, which had been cryopreserved at the time of a previous IVF attempt. Two days before the scheduled transfer, eight oocytes were thawed; 5/8 (63%) oocytes survived and 4/5 (80%) oocytes fertilized after intracytoplasmic sperm injection (ICSI) with the previously frozen epididymal spermatozoa. All four fertilized ova cleaved (100%). On day 2 after thawing, four embryos were transferred; three with two cells (grade II) and one with three cells (grade III). Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation, and the patient was delivered by Caesarean section at 40 weeks of gestation; the baby boy weighed 3025 g, and measured 51 cm, with Apgar of 10 in the 1st and 5th min. The cryopreservation and warming protocol used for this study yielded very favourable results, comparing well with reports in the literature. This case report demonstrates that it is possible to obtain high rates of oocyte survival following thawing and high rates of fertilization after ICSI, with viable development of the resulting embryos.

Chemical degradation of wastes of antineoplastic agents amsacrine, azathioprine, asparaginase and thiotepa.

As a part of a program devoted to the destruction of antineoplastic agents, three chemical methods readily available in the hospital environment, viz. oxidation with sodium hypochlorite (NaClO, 5%), hydrogen peroxide (H2O2, 30%), and Fenton reagent (FeCl2.2H2O; 0.3 g in 10 ml H2O2, 30%), were tested for the degradation of four anticancer drugs: Amsacrine, Azathioprine, Asparaginase and Thiotepa. The efficiency of the degradation was monitored by high-performance liquid chromatography. The mutagenicity of the degradation residues were tested by Ames test using tester strains Salmonella typhimurium TA 97a, TA 98, TA 100 and TA 102 with and without an exogenous metabolic activation system. Using sodium hypochlorite, 98.5% of Amsacrine, 99.0% of Azathioprine, 99.5% of Asparaginase and 98.7% of Thiotepa were destroyed after 1 hr. The hydrogen peroxide treatment destroyed 99% of Asparaginase and 98.7% of Thiotepa in 1 hr. However, this procedure was not efficient for the treatment of Amsacrine (28% after 16 hr) and of Azathioprine (53% degradation in 4 hr). The action of Fenton reagent resulted in the destruction of 98% of Amsacrine, and 99.5% of Azathioprine, 98.5% of Asparaginase and 98.7% of Thiotepa in 1 hr. In all cases where a high degree of degradation was achieved, the residues obtained weee non mutagenic.

Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with DNA adduction.

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, has been implicated as an etiologic agent in the Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Compared with unaffected individuals, patients suffering from BEN and/or urinary tract tumors were more frequently found to have a capacity for rapid debrisoquine (DB) metabolism, a metabolic reaction related mostly to cytochrome P450 (CYP) 2D in humans. Earlier studies, using female DA and Lewis rats phenotyped as poor or extensive DB metabolizers respectively, revealed a parallelism between DB-4 hydroxylation and OTA-4 hydroxylation. To investigate whether genetic polymorphism modifies tumor induction, we have compared both OTA-induced renal carcinogenicity and DNA adducts in DA and Lewis rats of both sexes. OTA induced renal adenocarcinoma, DA male rats being most responsive, while DA females were resistant. Lewis rats showed an intermediate renal tumor response. OTA also induced malignant transitional cell carcinomas of the bladder in DA male rats only. DNA adducts in the kidney, as judged by the nature of spots and prevalence in OTA-treated animals, were significantly correlated with renal carcinogenicity of OTA, being highest in DA males and lowest in DA females. A parallelism between karyomegalies and tumors of the kidney was observed. In conclusion, our results classify OTA as a genotoxic carcinogen in rats, with sex-specific response controlled in part by drug-metabolizing enzymes that convert OTA into reactive intermediates.

Chemical degradation of wastes of antineoplastic agents. 2: Six anthracyclines: idarubicin, doxorubicin, epirubicin, pirarubicin, aclarubicin, and daunorubicin.

OBJECT: Handling of genotoxic compounds commonly used in cancer chemotherapy generates contaminated wastes that require decontamination before disposal. Chemical methods are an alternative and/or a complement to incineration for the treatment of wastes and spills. METHODS: As part of a program initiated by the International Agency for Research on Cancer (IARC), 3 chemical methods readily available in the hospital environment--sodium hypochlorite (NaOCl, 5.25%), hydrogen peroxide (H2O2, < or = 30%) and Fenton reagent (FeCl2, 2H2O; 0.3 g in 10 ml H2O2, 30%)--are being tested for the degradation of a total of 32 antineoplastic agents. The efficiency of degradation was monitored by high-pressure liquid chromatography. The mutagenicity of the degradation residues were tested by the Ames test using tester strains Salmonella typhimurium TA 97a, TA 98, TA 100, and TA 102 with and without an exogenous metabolic activation system. RESULTS: The first results obtained for the degradation of cyclophosphamide, ifosfamide, and melphalan have been published in this journal. The present manuscript reports the results of the investigation of a series of six anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, and pirarubicin) commonly used in chemotherapy treatment. Pharmaceutical preparations corresponding to the most concentrated administration solutions in either NaCl (0.9%) or dextrose (5%) were inactivated by oxidation volume/volume with each of the methods for at least 1 h. Complete degradation into nonmutagenic residues of all the tested compounds was observed after 1 h for the NaOCl (5.25%) treatment as previously reported for the first study. CONCLUSION: Sodium hypochlorite (5.25%) is an efficient reagent for the chemical degradation of the nine drugs tested thus far.

Characterization of the cytochrome P450 isozyme that metabolizes ochratoxin A, using metabolic inducers, inhibitors and antibodies.

The phenotypic pattern of drug biotransformation is determined by both host and environmental factors. Debrisoquine is a good probe for phenotyping individuals, as its metabolism is not affected by age, gender, smoking habits or alcohol intake. People with Balkan endemic nephropathy or with urinary tract tumours in endemic areas are more frequently extensive metabolizers of debrisoquine than are healthy people. This finding has led to studies of the possible relationship between the metabolism of ochratoxin A and its toxicity and carcinogenicity on experimental models. Ochratoxin A is metabolized mainly in the liver into R- and S-isomers of 4- and 10-hydroxyochratoxin A, and the reaction is catalysed by cytochrome P450 haemoprotein. Animal species that are genetically different in their capacity to metabolize debrisoquine differ similarly in their capacity to metabolize ochratoxin A: female DA rats that are poor metabolizers of debrisoquine also poorly metabolize ochratoxin A, as assayed by urinary excretion of both the parent compound and of 4-hydroxyochratoxin A. Ochratoxin A hydroxylase activity is low in DA rat liver (and kidney) but is inducible by phenobarbital and 3-methylcholanthrene; debrisoquine hydroxylase is not known to be inducible by enzyme inducers. The reaction of ochratoxin A hydroxylase thus resembles those induced by 3-methyl-cholanthrene and catalysed by cytochrome P450IA. Ochratoxin A hydroxylase activity was further characterized in the livers of B6 and D2 mice that had been treated with typical enzyme inducers. Ochratoxin A hydroxylase was weakly inducible by phenobarbital, 3-methyl-cholanthrene and 2,4,7,8-tetrachlorodibenzodioxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Ochratoxin A and other mycotoxins in cereals from an area of Balkan endemic nephropathy and urinary tract tumours in Bulgaria.

The etiology of Balkan endemic nephropathy and urinary tract tumours in the rural population of the endemic regions remains unknown. As one hypothesis involves mycotoxins, a survey was carried out to investigate the possible involvement of the nephrotoxic mycotoxins ochratoxin A and citrinin. Recently, this survey was extended to screening for the presence of other mycotoxins--aflatoxins, citrinin, sterigmatocystin and zearalenone. A total of 524 samples of home-produced and home-stored beans and maize from the harvests of 1984, 1985, 1986, 1989 and 1990 were analysed. Ochratoxin A was found in samples from both endemic and nonendemic areas, but more of the samples from affected families were contaminated, and at higher levels, than those from unaffected households. Citrinin and aflatoxins B1 and G1 were also found more frequently in samples from endemic areas. These results support the theory that mycotoxins are involved in the etiology of Balkan endemic nephropathy and urinary tract tumours.

Polymorphic ochratoxin A hydroxylation in rat strains phenotyped as poor and extensive metabolizers of debrisoquine.

1. Dark agouti (DA) and Lewis rat strains, which show a genetic polymorphism for debrisoquine-4-hydroxylation, were treated either with a single dose of ochratoxin A (OA) or for 8 weeks with 5 doses per week. Levels of OA and its 4-hydroxy metabolite (4-hydroxy-OA) excreted in urine were determined. 2. At all doses, the metabolic ratio of OA:4-hydroxy-OA was two to five times greater in DA than in Lewis rats, as was the metabolic ratio of debrisoquine:4-hydroxy-debrisoquine. These results are consistent with our previous findings in vitro that hepatic and renal OA 4-hydroxylase activity is three to four times lower in DA than in Lewis rats. These data give further support to the possible co-segregation of genes regulating OA and debrisoquine 4-hydroxylation.

Oxidative destruction of hydrazines produces N-nitrosamines and other mutagenic species.

As part of the joint International Agency for Research on Cancer-National Cancer Institute program for the evaluation and development of methods for the degradation of chemical carcinogens, four oxidative techniques for the degradation of hydrazines were investigated. The oxidizing agents used were as follows: sodium hypochlorite, calcium hypochlorite, potassium iodate, and potassium permanganate in sulfuric acid. In each case, at least 99% of the hydrazine initially present was destroyed; however, the potential usefulness of these methods was compromised by the formation (in some reaction mixtures) of carcinogenic N-nitroso compounds and/or unknown mutagenic species. Oxidative degradation of hydrazines is recommended only for the decontamination of glassware and for the treatment of spills, for which reductive degradation methods are not suitable.

Interstrain comparison of hepatic and renal microsomal carcinogen metabolism and liver S9-mediated mutagenicity in DA and Lewis rats phenotyped as poor and extensive metabolizers of debrisoquine.

Lewis (extensive metabolizers) and DA (poor metabolizers) rat strains show genetic polymorphism for the gene regulating debrisoquine 4-hydroxylation in a manner analogous to human drug polymorphism and show also a different toxicological response to aflatoxin B1. In order to investigate the underlying mechanism(s) of the different drug-metabolizing capacities, the in vitro metabolism and liver S9-mediated mutagenicity of several drugs and carcinogens were studied in female Lewis and DA rat strains, using untreated, 3-methylcholanthrene-treated, and phenobarbital-treated animals. S9-mediated mutagenicity of aflatoxin B1 and hepatic and renal ochratoxin A 4-hydroxylase activity were much lower in DA rats; however, the activity of a number of other hepatic and renal drug-metabolizing enzymes did not show any interstrain difference. Slight strain differences were found in the inducibility of several hepatic drug-metabolizing enzymes, except for ochratoxin A 4-hydroxylase activity; the latter was inducible only by 3-methylcholanthrene in the DA strain and appeared to be linked genetically with other Ah locus-associated monooxygenases. Our data suggest that ochratoxin A 4-hydroxylase activity, which is low in the DA strain, is catalyzed by a cytochrome P-450 isozyme different from that responsible for debrisoquine 4-hydroxylation. Our results provide some insight into why the two metabolic oxidation phenotypes show different susceptibility to cancer induction and to the toxicity of certain environmental carcinogens, such as aflatoxin B1 and ochratoxin A.

Destruction of aromatic amines in laboratory wastes through oxidation with potassium permanganate/sulfuric acid into non-mutagenic derivatives.

Nine aromatic amines, i.e., benzidine; o-tolidine; o-dianisidine; 3,3'-dichlorobenzidine; 4-aminobiphenyl; 1- and 2-naphthylamine; 4,4'-methylene bis(2-chloroaniline) and m-toluenediamine, were oxidized with potassium permanganate/sulfuric acid. Experimental conditions for complete degradation of these aromatic amines are described. The disappearance of the parent compound through oxidation was measured using HPLC coupled with UV spectrophotometry. The corresponding degradation products were found to be non-mutagenic to Salmonella typhimurium strains TA100, TA98 and TA97, both in the presence and absence of a rat liver S9 activation system. A collaborative study, involving 11 laboratories, has shown the applicability and the reproducibility of this degradation method.

Destruction of carcinogenic and mutagenic N-nitrosamides in laboratory wastes.

The chemical degradation of five N-nitrosamides used widely for the experimental induction of cancer has been studied with the goal of identifying, and experimentally validating, reliable methods that can be recommended for the destruction of carcinogenic N-nitrosoureas and related compounds in laboratory wastes. Although data are not yet complete, preliminary evidence indicates that none of the five methods studied thus far is ideal for hazard-control purposes. Decomposition with 1 mol/L potassium hydroxide solution destroyed the N-nitrosamides, but generated diazoalkanes, which are carcinogenic, toxic and potentially explosive. Treatment with strong acid in the presence of sulfamic acid or iron filings completely decomposed all N-nitrosamides without forming diazoalkanes, but failed in the presence of solvents which were immiscible with water. Cleavage with hydrogen bromide in glacial acetic acid proceeded to a point of maximum degradation, following which gradual reformation of the N-nitrosamide was observed; this resynthesis could be avoided by carefully bubbling nitrogen through the reaction mixture, but degradation was slow or failed completely in the presence of hydroxylic solvents. Permanganate oxidation was effective in sulfuric acid solution, but was incomplete when an alcohol or dimethyl sulfoxide was present. Salmonella typhimurium tester strains TA1535, TA1530 and TA100, which detect base-pair substitutions in DNA, detected mutagenic degradation products in each of the destruction methods, with the exception of the hydrobromic acid/acetic acid procedure.

Inhibitory effect of betel nut extracts on endogenous nitrosation in humans.

Extracts of betel nut (Areca catechu) were tested for their capacity to inhibit the endogenous formation of nitrosamines by measurement of the amount of urinary N-nitroso-L-proline (NPRO) following ingestion of sodium nitrate (300 mg) and L-proline (300 mg) by 2 volunteers. A water extract of the dried nuts, an ether extract containing mainly (+)-catechin and (-)-epicatechin, and a caffeine-precipitated n-butyl alcohol extract containing primarily proanthocyanidins (tannins) strongly reduced the endogenous formation of NPRO. An average of 14.7 and 10.9 micrograms NPRO (8 expts per individual) was excreted in the urine of the 2 volunteers over a 24-hour period following the intake of sodium nitrate and L-proline. The water extract and the proanthocyanidin (tannin)-containing extract, both of which contain the dose equivalent of one-quarter of a nut, reduced the excreted NPRO to background levels, which varied from 0.5 to 3.6 micrograms and from 0.6 to 2.1 micrograms (6 expts) in 24-hour urine samples from the 2 volunteers. These results may exemplify the way in which naturally occurring phenolics, which are ingested daily in relatively large quantities, could affect the endogenous formation of carcinogenic nitrosamines.

Limitations in protection afforded by gloves in laboratory handling of aflatoxins.

The permeability of latex and vinyl laboratory gloves by aflatoxins in chloroform or dimethyl sulfoxide (DMSO) has been investigated. Latex gloves provide good protection against permeation by aflatoxins in DMSO, but aflatoxins in chloroform permeate all types of laboratory gloves. If gloves are contaminated when aflatoxins in chloroform are handled, an immediate change of gloves is recommended.