Absence Of Dimethyl Sulfoxide Research Articles

Mechanism of inhibition of the Ca(2+)-ATPase by spermine and other polycationic compounds.

The ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of polyamines, including spermine, spermidine, and poly(arginine). The effects of spermine on the ATPase are highly specific. It has no effect on the affinity of the ATPase for Ca2+ or ATP, and no effect on the rate of phosphorylation by ATP. When the ATPase is phosphorylated with Pi in the presence of dimethyl sulfoxide at pH 6.0, and then dephosphorylation is induced by dilution in buffer at pH 7.5 in the absence of dimethyl sulfoxide, spermine is found to have no effect on the rate of dephosphorylation. If the ATPase is phosphorylated with [gamma-32P]ATP and the rate of loss of radiolabeled phosphoenzyme is measured following the addition of unlabeled ATP, spermine is found to decrease the rate of loss of radiolabel, consistent with an effect of spermine on the rate of the Ca2E1P-->E2P step. Direct measurement confirms that spermine decreases the rate of dissociation of Ca2+ from the phosphorylated ATPase (Ca2E1P-->E2P), with the decrease in the rate of this step explaining the inhibition of ATPase activity. Spermine also increases the equilibrium constant E1/E2 and inhibits phosphorylation of the ATPase by Pi by competition with the Mg2+ essential for the reaction. It is suggested that spermine could bind to the site on the Ca(2+)-ATPase that interacts with phospholamban.

The effect of dimethylsulfoxide on the tegumental brush border of the cestode Hymenolepis diminuta.

Dimethyl sulfoxide (DMSO), commonly used for cryoprotection or for the solvation of cytoskeleton-modifying drugs, causes changes in the topology of the plasma membrane of the tegumental brush border in the tapeworm Hymenolepis diminuta; however, relatively long exposures of high concentrations are required. In tapeworms treated with DMSO concentrations of greater than or equal to 1% in the present study, the interaction of the tegumental surface membrane with the underlying cytoskeleton may have been disrupted at focal points in the brush border, resulting in a partial loss of the membrane anchoring required for the structural integrity of the brush border. Blebbing of the tegumental surface was prominent only after exposure to 1% DMSO for 20 h in in vitro culture with RPMI 1640, and vesiculation of the membrane along the microvillar (microtriche) shafts, which may have been related to the in vitro conditions, was amplified by the presence of concentrations of greater than or equal to 1% DMSO in the incubation medium. The tegumental response to DMSO was not uniform but regional, consistently appearing to be more prevalent on the distal aspects of each proglottid rather than on the edge proximal to the scolex. Blebbing and vesiculation were not seen on the basal aspect of the tegument, including the basal ectocytoplasmic plasma membrane, the perikarya, and the internuncial processes. Microvillar core bundles of actin microfilaments persisted following 8 h in vitro exposure to all three concentrations of DMSO tested (0.1%, 1%, 5%); however, only in tapeworms that were treated in vitro with 5% DMSO for greater than or equal to 8 h did core microfilament bundles appear to lose the rigidly straight and parallel organization characteristic of control tapeworms that were incubated either in the absence of DMSO or with 0.1% DMSO. Other components of the brush border cytoskeleton (i.e., microvillar caps, junctional tubules, and tunics) appeared unaffected by DMSO except at foci where blebbing occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dimethylsulfoxide on the proliferation and glycosaminoglycan synthesis of rat prostate adenocarcinoma cells (PAIII) in vitro: Isolation and characterization of DMSO‐resistant cells

Dimethylsulfoxide (DMSO) modulates the tumorigenicity and other characteristics of some malignant cell lines in vitro. We have investigated DMSO effects on cell proliferation and glycosaminoglycan (GAG) synthesis in rat prostate adenocarcinoma (PAIII) cells in culture. DMSO inhibited cell proliferation and GAG synthesis and shedding. Cells that survived the initial exposure to 2.5% DMSO could be propagated in this concentration of the agent and were designated PAIII-DMSO resistant (PAIII-DMSOr). PAIII-DMSOr cells showed reversible indications of increased differentiation such as decreased growth rate and saturation density. Although the PAIII-DMSOr cells were grown in 2.5% DMSO, they had normal or elevated GAG content. The major GAG of both PAIII and PAIII-DMSOr cells was undersulfated heparan sulfate. Verapamil, a calcium channel blocker that reverses drug resistance in tumor cells, stimulated the growth of PAIII-DMSOr cells in the presence of 2.5% DMSO, but was inhibitory in the absence of DMSO. Growth of PAIII cells was inhibited by the differentiating agents sodium butyrate and retinoic acid and by the ionophore monensin. Interestingly, growth of PAIII-DMSOr cells in the presence of 2.5% DMSO was largely unaffected by sodium butyrate or retinoic acid. The results suggest that (1) PAIII-DMSOr cells arise from the induction of a compensation mechanism to DMSO effects in a preexisting population of cells: (2) there is a correlation between GAG synthesis and cell proliferation; and (3) further study of the verapamil effect may help elucidate the mechanism of the DMSO-induced differentiation of cancer cells.

Radioprotective Effects of Dimethyl Sulfoxide in Golden Hamster Embryo Cells Exposed to γ Rays at 77 K: II. Protection from Lethal, Chromosomal, and DNA Damage

Golden hamster embryo cells were exposed to 137Cs gamma rays in the presence or absence of dimethyl sulfoxide at both 310 and 77 K. Dimethyl sulfoxide gave significant protection against cell killing at both 310 and 77 K. The extent of radioprotection with 1.28 M dimethyl sulfoxide at 77 K was 85-89% of the lethal effects observed in the absence of dimethyl sulfoxide at 310 K; the dose-modifying factor was 5.7. Dimethyl sulfoxide also exerted protected against gamma-ray-induced DNA single-strand breaks and chromosomal aberrations with a maximum protection of 80-100% at a dimethyl sulfoxide concentration of 1.28 M at 77 K. At 77 K, H atoms, ion holes, and electrons can migrate through frozen cells but OH radicals cannot diffuse. Thus the protective effects of dimethyl sulfoxide against cell killing, chromosomal aberrations, and DNA single-strand breaks at 77 K may be due to the scavenging of H atoms or other ions, rather than OH radicals.

Topical β-aminopropionitrile and biochemistry of granuloma tissue

The changes of connective tissue in granuloma development were studied up to 20 days following subcutaneous implantation of polyvinyl alcohol (PVA) sponges in rats. The total protein and collagen content were found to increase from the 4th to 12th days of implantation while the content of DNA continuously decreased with the age of the granuloma. Administration of β-aminopropionitrile (βAPN) fumarate on the intact skin overlying the implanted PVA sponge in the presence and absence of dimethyl sulfoxide (DMSO) during the growth phase of granuloma development (4th through 12th days after implantation) was accompanied by significant inhibition of collagen polymerization. This was documented by a significant increase of collagen extractable into 1 N NaCl, by a significantly reduced content of aldehydes in collagens extractable into neutral salt solution, and by a significant increase of α subunits and α/β ratio. Topical βAPN administration had no effect on the content of DNA, noncollagenous proteins, or total hydroxyproline. We conclude that topically administered βAPN fumarate onto the intact skin penetrates the stratum corneum and effectively inhibits collagen crosslinking in the underlying granuloma tissue. The extent of the effectiveness was found to be significantly greater in the presence of DMSO.

Antioxidants protect cultured bovine lung endothelial cells from injury by endotoxin.

Endotoxin injures bovine pulmonary endothelial cells in culture but the cytotoxicity is unaffected by a host of antiinflammatory drugs. We hypothesized that agents which could decrease intracellular concentrations of toxic metabolites of O2 would prevent endotoxin effects on cultured pulmonary artery endothelial cells. We measured endotoxin-induced release of lactate dehydrogenase (LDH) from and production of prostanoids by cultured bovine pulmonary endothelial cells in the presence and absence of dimethyl sulfoxide (DMSO) and the xanthine oxidase inhibitor allopurinol. Escherichia coli endotoxin (0.001-10 micrograms/ml) caused a dose-related release of LDH and stimulated production of both prostacyclin [measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] and prostaglandin E2 (PGE2). Both DMSO and allopurinol decreased endotoxin-induced LDH release; this effect was related to concentration of the drugs (0-2% for DMSO and 0-0.3 mg/ml for allopurinol). Both drugs also prevented endotoxin-induced changes in endothelial morphology. Endotoxin increased intracellular reduction of the redox dye nitro blue tetrazolium, caused intracellular oxidation of 2',7'-dichlorofluorescein diacetate and caused release of conjugated dienes from endothelial cells; both DMSO and allopurinol inhibited those responses. DMSO, but not allopurinol, prevented endotoxin-induced production of prostacyclin and PGE2 by endothelium. Direct injury of pulmonary endothelium by endotoxin is inhibited by two chemically dissimilar drugs which have a common potential for decreasing intracellular concentrations of toxic metabolites of O2; indirect evidence suggests that potential as a mechanism for the protective effects of the drugs.

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide.

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium Pi by mitochondrial F1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium Pi in equilibrium H18OH exchange and of the dependence of formation of enzyme-bound ATP on Pi concentration. Attainment of an apparent equilibrium between medium Pi and bound ATP requires longer than 30 min, even though the rates of Pi binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each Pi that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and Pi is much slower than that in the absence of dimethyl sulfoxide as measured with sufficiently low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and Pi is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of Pi to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and Pi.

Differential Effects of Radical Scavengers on X-Ray-Induced Mutation and Cytotoxicity in Human Cells

The cytotoxic and mutagenic effects of X irradiation on a human lymphoblast cell line were examined in the presence of two radioprotective agents which modulate damage to DNA. The cells were treated with X rays alone or in the presence of either dimethyl sulfoxide or cysteamine. Surviving fraction and mutation to trifluorothymidine resistance (tk locus) and to 6-thioguanine resistance (hgprt locus) were measured. Survival was enhanced when the cells were irradiated in the presence of dimethyl sulfoxide; the D0 rose from 58 to 107 rad. However, at both genetic loci the induced mutant fractions were identical in the presence or absence of dimethyl sulfoxide. Survival was enhanced to a greater degree when the cells were irradiated in the presence of cysteamine; the D0 rose from 58 to 200 rad. Cysteamine also protected the cells from X-ray-induced mutation; the frequencies of X-ray-induced mutation at both the tk and hgprt loci were reduced by 50-75%. No protective effects were observed unless dimethyl sulfoxide or cysteamine was present during irradiation. These findings are discussed in terms of the hypothesis that, unlike for cell killing, radiation-induced mutagenesis in human lymphoblast cells is not mediated by the actions of aqueous free radicals, but rather by the direct effects of ionizing radiation.

Fixation of radiation-induced potentially lethal damage by anisotonic treatment and its modification by DMSO or BrdUrd in V79 cells.

The effects of radiosensitization by bromodeoxyuridine (BrdUrd) substitution and radioprotection by dimethyl sulfoxide (DMSO) have been examined in relation to fixation and repair of radiation damage by anisotonic treatment. The fixation of radiation damage in cells exposed to 0.05 M or 1.5 M NaCl after irradiation was the same at equal survival levels irrespective of (BrdUrd) incorporation into the DNA. Also, during incubation between irradiation and a subsequent anisotonic treatment, cells containing BrdUrd repaired radiation damage to the same extents as cells without BrdUrd. DMSO treatment resulted in radioprotection. Fixation, by anisotonic salt treatment, of damage resulting from irradiation in the presence of DMSO was less extensive than from irradiation in the absence of DMSO, even though X-ray doses were adjusted to give equal survival levels. Recovery during incubation at 37 degrees C between irradiation and a subsequent salt treatment occurred for irradiation in the presence and absence of DMSO. These data show that the alteration of DNA radiosensitivity by BrdUrd had no effect on fixation or repair of radiation damage as assessed by salt treatment, while DMSO which is an OH scavenger caused the damage to be less susceptible to fixation and this damage was repaired during incubation at 37 degrees C.

Mechanism of inactivation of the polyene antibiotic amphotericin B. Evidence for radical formation in the process of autooxidation.

Radical formation during autooxidation of the polyene antibiotic amphotericin B was monitored by following the kinetics of decay of the ESR signal of a stable nitroxide. The kinetics were seen to depend both on antibiotic and on nitroxide concentration. The radicals formed were studied by spin trapping. Three preparations--clinical Fungizone, amphotericin B suspended in buffer, and amphotericin B in buffer - 10% dimethyl sulfoxide--yielded spin adducts of different nature and/or concentrations. In the absence of dimethyl sulfoxide, amphotericin B yielded at least two carbon-centered radicals whose spectra indicated restricted mobility. This suggests that the radicals arise from the whole amphotericin B molecule located in molecular aggregates present in the preparations, and not from smaller and possibly more water-soluble fragments of the antibiotic. In the presence of dimethyl sulfoxide the spin adducts derived from secondary carbon radicals originated from this solvent. Their spectra were indicative of fast tumbling. Direct evidence for autooxidation was obtained by measuring oxygen consumption. All processes examined occurred at the same time scale observed for drug inactivation, in agreement with the idea that loss of activity is due to antibiotic autooxidation.

A model reaction of the mitochondrial ATP synthesis.

Mitochondrial F1-F0 complex catalyzes ATP synthesis coupled with proton translocation down the electrochemical potential gradient (ΔμH+) across the inner membrane. F1, the catalytic sector of the complex, can be detached from the membrane to be a soluble protein.Recently, we found that soluble F1 catalyzed ATP synthesis from medium ADP and Pi in the presence of dimethylsulfoxide (DMSO). This finding indicates that energy input of ΔμH+ is not essential to covalent binding of ADP and Pi to form ATP on F1. The synthesized ATP was bound to F1 to compose F1-ATP complex, suggesting that the energy input is necessary for the release of ATP from F1.The F1-ATP complex seemed to be formed also in the absence of DMSO. DMSO increased the affinity of F1 for Pi and shifted the equilibrium between F1-ATP complex and F1-ADP-Pi complex.Since it is the most simple ATP-synthesizing system ever known, it might be utilized to solve other problems about the mechanism of ATP synthesis. F1 has two types of nucleotide-binding sites, tight binding sites and exchangeble binding sites. However, it has not been exclusively determined which is the catalytic site of ATP synthesis. We found that ATP was formed by soluble F1 from ADP bound to the exchangeable binding site (s) but not from that bound to the tight binding sites.

Facile dehydrobromination ofvic‐dibromo fatty acids: A one‐vessel bromination‐dehydrobromination of oleic acid to stearolic acid

AbstractThe effects of time and temperature on the dehydrobromination by alcoholic KOH of 9,10‐dibromostearic acid to 9(10)‐bromo‐9‐octadecenoic and stearolic acid (9‐octadecynoic acid) have been studied. Dimethylsulfoxide (DMSO) in n‐propanol‐KOH solution has been found to be an effective cosolvent‐catalyst. In the absence of DMSO, the first monodehydrobromination step to the ethenylene bromide is quantitative in 5 min at 60 C and the second dehydrobromination step to the acetylenic acid is complete in about 6 hr at 100 C. In the presence of DMSO, the dehydrobromination to stearolic acid is quantitative at 100 C in 1–2 hr. A 1‐vessel oleic acid bromination‐dehydrobromination has been devised to give stearolic acid in 98% yield and purity.

Changes in the expression of the alpha alpha form of enolase during neuroblastoma differentiation.

The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form alpha alpha, and an increase in the activity associated with the gamma-containing isozymes (alpha gamma plus gamma gamma); in the absence of DMSO, there is no decrease in alpha alpha or in total enolase activity. In order to study the mechanism of the changes in alpha alpha, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the alpha antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the alpha antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the alpha antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t1/2 greater than 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the alpha subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the alpha gene that occurs in vivo during neuronal differentiation.

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.

Morphologic effect of dimethyl sulfoxide on the blood-brain barrier.

Dimethyl sulfoxide (DMSO) opens the blood-brain barrier of mice to the enzymatic tracer horseradish peroxidase. A single injection of horseradish peroxidase in 10 to 15 percent DMSO into the tail vein along with 10 to 15 percent DMSO delivered intraperitoneally allowed horseradish peroxidase to fill the extracellular clefts throughout the brain within 2 hours. In the absence of DMSO, peroxidase failed to enter brain parenchyma except through the circumventricular organs. Opening of the blood-brain barrier by DMSO is reversible. Dimethyl sulfoxide stimulated the pinocytosis of horseradish peroxidase by the cerebral endothelium; the peroxidase was then directed to lysosomal dense bodies for degradation. Vesicular transport of horseradish peroxidase from the luminal to the abluminal wall of the endothelial cell was not observed. Dimethyl sulfoxide did not alter the morphology of endothelial cells or brain parenchyma.

Effect of dimethylsulfoxide on two synthetic Asn-X-Thr containing substrates of the oligosaccharyltransferase

The enzymatic transfer of oligosaccharides from oligosaccharide lipids to synthetic Asn-X-Thr containing peptides of various length was studied in presence and absence of dimethylsulfoxide. Up to 13.5%, this solvent was found to specifically enhance the efficiency of a ribonuclease heptapeptide to be substrate of the thyroid oligosaccharyltransferase and in contrast, not that of a tripeptide. Circular dichroic analysis performed in various dimethylsulfoxidewater mixtures showed that only the heptapeptide underwent conformational modifications when increasing the concentration in dimethylsulfoxide. Some ordered structure in the immediate vicinity of the Asn-X-Thr signal sequence thus appears of importance in the N-glycosylation process.

The mutagenic propeeties of hexachloroacetone in short-term bacterial mutagen assay systems

Hexachloroacetone, CCl 3-CO-CCl 3, reverts the Ames strains TA98 and TA-100 but not the non-plasmid strains TA1537, TA1535 and TA1538. In the absence of solvent, the number of revertant colonies is 5 times the spontaneous reversion rate for TA100 and 10 times the spontaneous reversion rate for TA98 with 26 mg hexachloroacetone per plate. This effect is seen in the absence of rat liver microsomes. In dimethylsulfoxide (DMSO) solution a more complicated pattern is seen. In DMSO solution cooled between 18 and 20°C, the maximum number of revertants is similar to that found in the absence of DMSO, but only 1.75 mg hexachloroacetone per plate is needed. When DMSO solution of hexachloroacetone is warmed above 20°C, a yellow color develops and the solution becomes more toxic to the test bacteria. The maximum number of revertants is then produced at about 0.5 mg hexachloroacetone per plate. Hexachloroacetone is found to be active, without microsomal activation, in the E. coli WP-2 and E. coli rec-BC test systems. Hexachloroacetone readily reacts with water in DMSO solutions to form the non-mutagenic hexachloroacetone hydrate.

Sperm number required for maximum fertility of chicken semen processed for freezing.

Two independent experiments were designed to determine the number of sperm needed for maximum fertility of chicken semen processed for freezing. Pooled semen samples were either diluted 1:2 or 1:5 with the Beltsville Poultry Semen Extender, while undiluted samples served as controls. Samples were then subsequently processed (pre-freeze), according to the Beltsville method, as if they were going to be frozen. At two stages of processing, after 2-hr holding at 5 degrees C in the absence of dimethylsulfoxide (DMSO) and after an additional 2-hr holding at 5 degrees C in the presence of 4 p. 100 DMSO, aliquots were removed and tested for fertility. Each test hen was intravaginally inseminated with either 100, 300, or 600 million sperm from undiluted or processed semen samples. The number of sperm required for the undiluted controls was determined to be 100 million sperm whereas the fertility of samples processed with 4 p. 100 DMSO was significantly lower than the undiluted controls regardless of the sperm number inseminated or the dilution rate. Dilution rate did influence the number of sperm required for samples cooled for 2 hrs at 5 degrees C in the absence of DMSO. In samples diluted 1:2, 100 million sperm were needed for maximum fertility, whereas 300 million sperm were needed with samples diluted 1:5.