Fertility of frozen sex-sorted sperm at 4 × 106 sperm per dose in lactating dairy cows in seasonal-calving pasture-based herds.

The objective was to evaluate the reproductive performance of frozen sex-sorted sperm at 4 × 106 sperm per dose (SexedULTRA 4M, Sexing Technologies, Navasota, TX) relative to frozen conventional sperm in seasonal-calving pasture-based dairy cows. Semen from Holstein-Friesian (n = 8) and Jersey (n = 2) bulls was used. Four of the Holstein bulls used were resident at or near a sex-sorting laboratory (Cogent, UK, or ST Benelux, the Netherlands). The remaining 6 bulls were located at studs in Ireland. For these 6 bulls, ejaculates were collected, diluted with transport medium, and couriered to Cogent in parcel shippers. Transit time from ejaculation to arrival at the sorting laboratory was 6 to 7 h. For all bulls, ejaculates were split and processed to provide frozen conventional sperm (CONV) at 15 × 106 sperm per straw and frozen sex-sorted (SS) sperm at 4 × 106 sperm per straw and used to inseminate lactating dairy cows after spontaneous estrus. Pregnancy diagnosis was performed by ultrasound scanning (n = 7,246 records available for analysis). Generalized linear mixed models were used to examine effects on pregnancy per AI (P/AI) at first artificial insemination, with sperm treatment (CONV vs. SS), bull (n = 10), and treatment × bull interaction as the fixed effects, and herd (n = 142) as a random effect. Overall, P/AI was greater for cows inseminated with CONV than for those inseminated with SS (59.9% vs. 45.5%; 76.0% relative to CONV). This study was not designed to compare resident bulls vs. shipped ejaculates, but the magnitude of the difference between P/AI achieved by CONV and SS was apparently less for resident bulls (60.3% vs. 50.2%) than for shipped ejaculates (58.6% vs. 40.7%). We discovered a treatment × bull interaction for shipped ejaculates (P/AI ranged from 45 to 86% relative to CONV) but not for the resident bulls (P/AI ranged from 81 to 87% relative to CONV). Relative P/AI of SS compared with CONV was greater in cows with high or average fertility potential (76.1% and 78.3%, respectively) than in cows with low fertility potential (58.1%). In 33.1% of the enrolled herds, the P/AI achieved with SS was 90% or more of the P/AI achieved with CONV; this was mainly explained by herds in which SS performed exceptionally well but CONV performed poorly. In conclusion, SS had lower overall P/AI compared with CONV; however, P/AI achieved with SS was dependent on the bull, fertility potential of the cow, and herd. Strategies to improve the P/AI with SS in seasonal-calving pasture-based lactating dairy cows require further research.

Evaluation of delayed timing of artificial insemination with sex-sorted sperm on pregnancy per artificial insemination in seasonal-calving, pasture-based lactating dairy cows.

The objective was to use ovulation synchronization with timed artificial insemination (TAI) to evaluate the effect of timing of artificial insemination (AI) with frozen sex-sorted sperm on fertility performance in pasture-based compact calving herds. Ejaculates from 3 Holstein-Friesian bulls were split and processed to provide frozen sex-sorted sperm (SS) at 4 × 106 sperm per straw, and frozen conventional sperm at 15 × 106 sperm per straw (CONV). A modified Progesterone-Ovsynch protocol was used for estrous synchronization, with TAI occurring 16 h after the second GnRH injection for cows assigned to CONV, and either 16 h (SS-16) or 22 h (SS-22) for cows assigned to SS. Pregnancy diagnosis was conducted by transrectal ultrasound scanning of the uterus 35 to 40 d after TAI (n = 2,175 records available for analysis). Generalized linear mixed models were used to examine the effects of treatment on pregnancy per artificial insemination (P/AI). Fixed effects included treatment (n = 3), bull (n = 3), treatment by bull interaction, parity (n = 4), days-in-milk category (n = 3), and treatment by days-in-milk category, with herd (n = 24) included as a random effect. Pregnancy per AI was greater for CONV compared with both SS-16 and SS-22 (61.1%, 49.0%, and 51.3%, respectively), and the SS treatments did not differ from each other (relative P/AI for SS-16 and SS-22 vs. CONV were 80.2% and 84.0%, respectively). There were significant bull and treatment by bull interaction effects. Additional analysis was undertaken using a model that included herd as a fixed effect. This analysis identified marked herd-to-herd variation (within-herd relative P/AI for the combined SS treatments vs. CONV ranged from 48-121%). The tertile of herds with the best performance achieved a mean relative P/AI of 100% (range = 91-121%), indicating that P/AI equivalent to CONV is achievable with SS. Conversely, the tertile of herds with the poorest performance achieved a mean relative P/AI of 67% (range = 48-77%). We found that SS resulted in poorer overall P/AI compared with CONV sperm regardless of timing of AI. Marked variation existed between herds; however, one-third of herds achieved P/AI results equal to CONV. Identification of factors responsible for the large herd-to-herd variation in P/AI with SS, and development of strategies to reduce this variation, warrant further research.

The relationship between anogenital distance and fertility, and genome-wide associations for anogenital distance in Irish Holstein-Friesian cows.

The evaluation of anogenital distance (AGD), the distance from the center of the anus to base of the clitoris, as a potential fertility trait for genetic selection in dairy cows has generated recent interest. The objectives of this cross-sectional observational study were to (1) characterize the distribution and variability of AGD, (2) determine factors associated with AGD, (3) estimate heritability for AGD, (4) identify single nucleotide polymorphisms (SNP) associated with phenotypic variation of AGD, and (5) validate the relationship between categories of AGD and fertility in Irish Holstein-Friesian cows. Anogenital distance was measured using digital calipers in 1,180 Holstein cows (mean ± standard deviation: 225 ± 79 d in milk) from 10 dairy herds located in Munster, Ireland. In addition, age (yr), weight (kg), height at hip (cm), and body condition score (BCS) at the time of AGD measurement were determined in a subset of 281 cows. Genotype information available from 908 cows was subsequently imputed to the Illumina Bovine High Density BeadChip (Illumina Inc., San Diego, CA) for genome-wide association analysis of phenotypic variation in AGD. Overall, AGD had a normal distribution and high variability (mean ± standard deviation; 119.2 ± 11.6 mm). Anogenital distance was weakly but positively associated with cow age, hip height, and body weight, and negatively associated with BCS; the phenotypic variation in AGD that was explainable by these variables was small (coefficient of determination; R2 = 0.09, 0.06, 0.10, and 0.02, respectively). The estimated heritability for AGD was 0.37 (standard error of mean ± 0.08). Six SNP of suggestive significance were identified on Bos taurus autosomes 6, 15, 20, and 26; however, none of these SNP was related to previously identified candidate genes for fertility. Cows were categorized into quartiles (Q1; 86 to 111 mm; n = 311, Q2; 112 to 120 mm; n = 330; Q3; 121 to 127 mm; n = 265, and Q4; 128 to 160 mm; n = 274) based on AGD and the association with reproductive outcomes examined (21-d submission rate, pregnancy to first AI, pregnancy rate within 21, 42 and 84-d after the farm mating start date, and number of times bred). None of the reproductive variables differed significantly between AGD categories. In summary, despite identification of high variability and moderate heritability for AGD in Irish Holstein-Friesian cows, reproductive outcomes did not differ between categories of AGD. This latter result differs from our previous finding of an inverse relationship between AGD and pregnancy outcomes in first- and second-parity Canadian Holstein cows, emphasizing the need to test and validate this new phenotype in diverse cow populations.

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function.

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen-thawed stallion semen.

The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100×106 spermatozoa mL-1 with 0.02mM vitamin E (VE) and 0, 1, 10 or 20ng mL-1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120min and viability, acrosome integrity and membrane fluidity were evaluated at 30min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20×106 spermatozoa mL-1 and stored at 4°C. After 1, 24, 48 and 72h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.

Differentiation and reversal of malignant changes in colon cancer through PPARgamma.

PPARgamma is a nuclear receptor that has a dominant regulatory role in differentiation of cells of the adipose lineage, and has recently been shown to be expressed in the colon. We show here that PPARgamma is expressed at high levels in both well- and poorly-differentiated adenocarcinomas, in normal colonic mucosa and in human colon cancer cell lines. Ligand activation of this receptor in colon cancer cells causes a considerable reduction in linear and clonogenic growth, increased expression of carcinoembryonic antigen and the reversal of many gene expression events specifically associated with colon cancer. Transplantable tumors derived from human colon cancer cells show a significant reduction of growth when mice are treated with troglitazone, a PPARgamma ligand. These results indicate that the growth and differentiation of colon cancer cells can be modulated through PPARgamma.

Tumor resistance to alkylating agents conferred by mechanisms operative only in vivo.

EMT-6 murine mammary tumors were made resistant to cis-diamminedichloroplatinum (II) (CDDP), carboplatin, cyclophosphamide (CTX), or thiotepa in vivo by treatment of tumor-bearing animals with the drug during a 6-month period. In spite of high levels of in vivo resistance, no significant resistance was observed when the cells from these tumors were exposed to the drugs in vitro. The pharmacokinetics of CDDP and CTX were altered in animals bearing the respective resistant tumors. The resistance of all tumor lines except for the EMT-6/thiotepa decreased during 3 to 6 months in vivo passage in the absence of drugs. These results indicate that very high levels of resistance to anticancer drugs can develop through mechanisms that are expressed only in vivo.

Review: Applications and benefits of sexed semen in dairy and beef herds.

The use of sexed semen in dairy and beef cattle production provides a number of benefits at both farm and industry levels. There is an increasing demand for dairy and beef products across the globe, which will necessitate a greater focus on improving production efficiency. In dairy farming, there is surplus production of unwanted male calves. Male dairy calves increase the risk of dystocia compared with heifer calves, and as an unwanted by-product of breeding with conventional semen, they have a low economic value. Incorporating sexed semen into the breeding programme can minimise the number of unwanted male dairy calves and reduce dystocia. Sexed semen can be used to generate herd replacements and additional heifers for herd expansion at a faster rate from within the herd, thereby minimising biosecurity risks associated with bringing in animals from different herds. Furthermore, the use of sexed semen can increase herd genetic gain compared with use of non-sorted semen. In dairy herds, a sustainable breeding strategy could combine usage of sexed semen to generate replacements only, and usage of beef semen on all dams that are not suitable for generating replacements. This results in increased genetic gain in dairy herd, increased value of beef output from the dairy herd, and reduced greenhouse gas emissions from beef. It is important to note, however, that even a small decrease in fertility of sexed semen relative to conventional semen can negate much of the economic benefit. A high fertility sexed semen product has the potential to accelerate herd expansion, minimise waste production, improve animal welfare and increase profitability compared with non-sorted conventional semen.

Novobiocin enhances alkylating agent cytotoxicity and DNA interstrand crosslinks in a murine model.

DNA-DNA crosslinks are the lethal cellular mechanism of bifunctional alkylating agent cytotoxicity. Novobiocin, an inhibitor of DNA topoisomerase II, impairs eukaryotic DNA repair of alkylating agent adducts and may increase the number of adducts and their resultant cytotoxicity in malignant cells. The effect of novobiocin on clonogenic survival and DNA crosslinking due to cisplatin (cDDP) and carmustine (BCNU) was studied. Novobiocin caused synergistic cytotoxicity in Chinese hamster ovary cells exposed to cDDP or BCNU. Novobiocin and cDDP increased the formation of DNA-DNA interstrand crosslinks six-fold greater than cDDP alone. The effect was schedule dependent. Novobiocin and cDDP or BCNU markedly reduced in vivo growth of a murine fibrosarcoma without increased host toxicity. As a modulating agent of cytotoxicity due to DNA-DNA crosslinking, novobiocin may enhance the clinical effectiveness of the alkylating agents in human cancer and offer insight into new therapeutic strategies.

Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality.

The aim of this study was to examine the effects of dietary supplementation with rumen protected n-6 or n-3 polyunsaturated fatty acids (PUFA) on the quantity and quality of semen from young post-pubertal dairy bulls. Pubertal Holstein-Friesian (n = 43) and Jersey (n = 7) bulls with a mean ± s.e.m. age and bodyweight of 420.1 ± 5.86 days and 382 ± 8.94 kg, respectively, were blocked on breed, weight, age and semen quality (based on the outcomes of two pre-trial ejaculates) and randomly assigned to one of three treatments: (i) a non-supplemented control (CTL, n = 15), (ii) rumen-protected safflower (SO, n = 15), (iii) rumen-protected n-3 PUFA-enriched fish oil (FO, n = 20). Bulls were fed their respective diets, ad libitum for 12 weeks; individual intakes were recorded using an electronic feeding system for the initial 6 weeks of the feeding period. Semen was collected via electro-ejaculation at weeks -2, -1, 0, 7, 10, 11 and 12 relative to the beginning of the trial period (week 0). On collection, semen volume, sperm concentration and progressive linear motility (PLM) were assessed. On weeks -2, -1, 0, 10, 11, 12, semen was packaged into 0.25 mL straws and frozen using a programmable freezer. On weeks -1, 7 and 11; a sub-sample of semen was separated into sperm and seminal plasma, by centrifugation and stored at - 20 °C until analysis of lipid composition. Semen from 10 bulls per treatment were used for post-thaw analysis at weeks 10, 11 and 12 (3 straws per ejaculate). Sperm motility was analysed by computer assisted semen analysis (CASA). In addition, membrane fluidity, acrosome reaction and oxidative stress were assessed using flow cytometry. Sperm from bulls fed SO had a 1.2 fold higher total n-6 PUFA content at week 11 compared to week -1 (P < 0.01) while bulls fed FO had a 1.3 fold higher total n-3 PUFA content, in sperm by week 11 (P < 0.01). There was no effect of diet on semen volume, concentration or PLM of sperm when assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls.

Relationship between in vitro sperm functional assessments, seminal plasma composition, and field fertility after AI with either non-sorted or sex-sorted bull semen.

The hypothesis of this study was that different in vitro parameters are required to predict the in vivo fertility of non-sorted (NS) and sex-sorted (SS) semen. Thus, the aim was to correlate in vitro bull sperm functional parameters (experiment 1) and seminal plasma composition (experiment 2) with pregnancy rates using 2 cohorts of bulls (NS and SS). Experiment 1: ejaculates from each bull (n = 3 ejaculates per bull; n = 6 bulls for both NS and SS) were assessed for motility, thermal stress tolerance and morphology using microscopy, and viability, osmotic resistance, mitochondrial membrane potential, and acrosome integrity using flow cytometry. Fertilizing ability was assessed using IVF. Experiment 2: ejaculates (n = 3 per bull; n = 8 and 6 bulls for NS and SS, respectively) were collected, seminal plasma harvested and frozen and later analyzed for amino acid and fatty acid composition using gas chromatography mass spectrometry. In the NS cohort of bulls, there was no correlation between pregnancy rate and any of the sperm functional parameters assessed. However, within the SS cohort, motility and viability were correlated with pregnancy rate (r = 0.84 and 0.80, respectively; P < 0.05). There was no correlation between IVF outcome and pregnancy rate in either the SS or NS cohort of bulls. In the NS cohort of bulls, concentrations of the amino acid isoleucine and the fatty acid tricosylic acid (C23:0) were correlated with pregnancy rate (r = 0.80 and 0.74, respectively; P < 0.05). Within the SS cohort of bulls, the amino acid glutamic acid and the fatty acid arachidic acid (C20:0) were correlated with pregnancy rate (r = 0.84 and 0.82, respectively; P < 0.05). In conclusion, this study suggests that different in vitro markers of fertility are required to predict the fertility of NS and SS sperm.

Effect of oxygen on the cytotoxicity and antitumor activity of etoposide.

The selective cytotoxicity of the epipodophyllotoxin etoposide toward normally oxygenated and hypoxic EMT6 mouse mammary tumor cells in culture was examined. Etoposide was much more toxic to normally oxygenated cells. The ratio (hypoxic to oxygenated) of drug concentrations producing 1 log of cell kill was approximately 30:1. Established FSa-11C fibrosarcomas of C3HeB/FeJ mice were treated with 10, 15, or 20 mg etoposide/kg body weight in a 6-day protocol. Fluosol-DA with or without breathing of carbogen (i.e., 95% O2-5% CO2) was added to the treatment program on days 1, 3, and 5. The combination of etoposide-Fluosol-DA-carbogen markedly enhanced tumor growth delay compared to the result with etoposide alone. The dose-modifying effect observed was 1.9 +/- 0.3. With the use of both single-dose and multiple-dose protocols for etoposide and Fluosol-DA with air or carbogen breathing, the survival of bone marrow cells was measured by colony formation in vitro (granulocyte-monocyte colony-forming units). Fluosol-DA and carbogen breathing did not increase the toxicity of etoposide to the bone marrow. Thus the enhancement in antitumor activity produced by the addition of Fluosol-DA and carbogen breathing to etoposide treatment was not accompanied by a concomitant increase in normal tissue toxicity and represents an increase in the therapeutic efficacy of etoposide.

Addition of misonidazole, etanidazole, or hyperthermia to treatment with fluosol-DA/carbogen/radiation.

The antitumor efficacy of adding the nitroimidazole radiosensitizing drugs misonidazole and etanidazole or hyperthermia (43 degrees C for 30 min) to Fluosol-DA/carbogen (95% O2/5% CO2) and irradiation was tested in the FSaIIC tumor system. Both the nitroimidazole drugs and hyperthermia produced additional tumor growth delays and tumor cell cytotoxicity when given with Fluosol-DA/carbogen, either before or after irradiation. For each of the modalities tested, the dose-modifying effect was greater when that therapy preceded rather than followed irradiation (misonidazole 2.7 vs. 1.9, etanidazole 2.4 vs. 1.7, hyperthermia 4.0 vs. 1.7 relative to the effect of radiotherapy alone). Because the nitroimidazole drugs must be present before radiation is administered to exert their radiosensitizing effect, the increase in tumor growth delay observed when these drugs cytotoxic to hypoxic cells were administered following Fluosol-DA/carbogen and irradiation suggests that Fluosol-DA/carbogen could not fully oxygenate the tumors and that the nitroimidazole drugs were effectively toxic to residual hypoxic cells. The treatment Fluosol-DA/carbogen----hyperthermia----irradiation produced a marked increase in tumor growth delay not seen with the sequence Fluosol-DA/carbogen----irradiation----hyperthermia. The results indicate that a treatment combination of radiation sensitizers may be more effective than irradiation plus Fluosol-DA with oxygen breathing alone.

Effect of various oxygenation conditions and fluosol-DA on cytotoxicity and antitumor activity of bleomycin in mice.

In an attempt to improve the antitumor efficacy of bleomycin, the effects of the oxygen-carrying emulsion Fluosol-DA and increased levels of inspired oxygen were tested in the mouse FSaIIC fibrosarcoma system. The dose-dependent cytotoxicity of bleomycin toward the FSaIIC cells in vitro was significantly decreased under hypoxic conditions, but it increased in a 95% O2-5% CO2 (carbogen) atmosphere as compared with the cytotoxicity of bleomycin in normally oxygenated cells. Investigations on the FSaIIC tumor in vivo also demonstrated that growth delays induced by bleomycin (10 mg/kg ip given on days 6, 10, 13, and 16) were significantly increased when one of the following treatments was given with each bleomycin injection: carbogen breathing for 2 hours (4.7 days), carbogen breathing for 6 hours (5.7 days), and breathing 3 atm of hyperbaric oxygen (6.3 days) versus normal air (3.3 days). When Fluosol-DA (12 mL/kg iv) was administered just before each bleomycin injection, the following growth delays were produced: 4.8 days with air breathing, 14.6 days with carbogen breathing for 2 hours, 14.9 days with carbogen breathing for 6 hours, and 19.7 days with breathing 100% O2 at 3 atm for 1 hour. Excision studies on the FSaIIC tumor also demonstrated that the cytotoxicity increased approximately fivefold when Fluosol-DA and carbogen breathing for 2 hours were combined with a single treatment with 10 mg of bleomycin/kg. In contrast, no measurable bone marrow toxicity was evident with this combined regimen. These results suggest that the use of Fluosol-DA plus carbogen breathing could add substantially to the clinical antitumor effects of bleomycin.

Enhancement of alkylating agent activity by SR-4233 in the FSaIIC murine fibrosarcoma.

BACKGROUND: The most commonly used antineoplastic drugs are more cytotoxic toward normally oxygenated tumor cells than toward hypoxic tumor cells. PURPOSE AND METHODS: To examine the ability of SR-4233, a new cytotoxic agent, to overcome the resistance of hypoxic tumor cells to antitumor alkylating agents, we tested the cytotoxic effect of SR-4233 alone and in combination with varying doses of cisplatin (CDDP), cyclophosphamide (CPM), carmustine (BCNU), or melphalan (L-PAM) on tumor cells and bone marrow cells isolated from C3H/FeJ mice bearing the FSaIIC fibrosarcoma. RESULTS: When SR-4233 alone was given, tumor cell killing was limited. When SR-4233 was administered just before single-dose treatment with CDDP, CPM, BCNU, or L-PAM, however, marked dose enhancement leading to increased cytotoxic effects on tumor cells and on bone marrow cells was observed. Similar experiments with tumor cell subpopulations, selected by Hoechst 33342 dye diffusion, confirmed that while cytotoxicity to both bright (oxygenated) and dim (hypoxic) cells was increased by combining each alkylating agent with SR-4233, the enhancement of the effect was relatively greater in the subpopulation of dim cells. The delay in the growth of tumors in animals treated with the combination of SR-4233 and CDDP, CPM, or L-PAM was 1.6-fold to 5.3-fold greater than that in animals treated with each alkylating agent alone. CONCLUSION: Our results suggest that SR-4233 may have the potential to improve the clinical efficacy of commonly used antitumor alkylating agents.

Combined modality therapy with bleomycin, hyperthermia, and radiation.

In an attempt to develop better combination therapies for use with local radiation, the interaction between bleomycin and hyperthermia +/- radiation was studied in the FSaIIC tumor system. In cells exposed in vitro to bleomycin at 37 degrees C and at pH 7.40, the drug was substantially more toxic toward normally oxygenated than hypoxic cells. At hyperthermic temperatures (42 degrees or 43 degrees C), however, the differential killing between the normally oxygenated and hypoxic cells disappeared and bleomycin became significantly more toxic. Exposure to bleomycin at pH 6.45 did not substantially alter the cytotoxicity of the drug at 42 degrees or 43 degrees C. In tumor growth delay experiments, combining bleomycin, hyperthermia, and radiation induced long delays, and the more successful sequences were bleomycin----radiation----hyperthermia or bleomycin----hyperthermia----radiation. If radiation was given prior to drug and hyperthermia, however, the sequence was significantly less effective. In tumor excision experiments performed 24 h after treatment, increasing doses of bleomycin produced a shallow, log-linear increase in tumor cell kill at 37 degrees C, but bleomycin followed by hyperthermia (43 degrees C, 30 min) led to about 1 log more cell killing. Administration of bleomycin just prior to treatment with a single dose of radiation was cytotoxically additive. In this assay the most effective trimodality treatment sequence was bleomycin----hyperthermia----radiation. In tumor subpopulations defined by Hoechst 33342 dye staining, bleomycin at 37 degrees C was about two-fold more toxic toward the bright (presumably well-oxygenated) cells than toward the dim (presumably hypoxic) cell subpopulation. The addition of hyperthermia following bleomycin produced nearly a log more tumor cell killing in both the bright and dim tumor cells. The combination of bleomycin followed by hyperthermia and then radiation was at least additive in the bright cells and caused a large cell kill, but in comparison, there was marked sparing of the dim cells. These results indicate that treatment with bleomycin and hyperthermia in conjunction with radiation can add substantially to tumor cell killing. This combination is significantly less effective in the hypoxic than oxic tumor regions, however, in spite of in vitro data which demonstrate that the cytotoxicity of bleomycin at hyperthermic temperatures is not oxygen-dependent.

Approaches to defining the mechanism of enhancement by Fluosol-DA 20% with carbogen of melphalan antitumor activity.

Fluosol-DA with carbogen (95% oxygen and 5% carbon dioxide) breathing can increase the efficacy of melphalan. Addition of Fluosol-DA to treatment with melphalan leads to a greater increase in tumor growth delay under conditions of air breathing and carbogen breathing than does the fat emulsion Intralipid. The ability of melphalan to kill tumor cells increased with dose over the range of drug examined. At the lower doses of drug there is some increase in tumor cell killing seen with the addition of carbogen breathing or Fluosol-DA and air breathing; however, at the highest dose of the drug this difference disappeared. Throughout the melphalan dosage range examined there is approximately 1 log greater tumor cell kill observed with the addition of Fluosol-DA and carbogen breathing compared to the drug treatment alone. There was no significant difference in the survival of bone marrow cells under any of the treatment conditions. Fluosol-DA itself with air or carbogen breathing produced no detectable cross-links in DNA from tumors treated in vivo. The cross-linking factors for melphalan with air or carbogen breathing and for melphalan plus Fluosol-DA and air breathing were similar; when carbogen breathing was added to the treatment combination, the cross-linking factor increased almost 3-fold. When melphalan was dissolved in Fluosol-DA, the melphalan moved quickly into the lipophilic perfluorochemical particles so that after 1 h 60% of the drug was in the perfluorochemical layer. At 24 h, 85-90% of the melphalan was sequestered in the perfluorochemical particles. The pharmacokinetics of [14C]melphalan alone, [14C]melphalan plus Fluosol-DA, and [14C]melphalan prepared in Fluosol-DA were studied in several tissues of FSaIIC fibrosarcoma-bearing mice. In general, the tissue absorption and distribution t1/2s for melphalan were shortened in the presence of Fluosol-DA (except for kidneys). Shifting the t1/2s for absorption and distribution to shorter times produces a much sharper and earlier peak in the drug exposure of the tumor. Fluosol-DA provides a relatively nontoxic means of increasing oxygen delivery to tumors and a therapeutically meaningful way of improving melphalan antitumor activity.

Effects of various oxygenation conditions on the enhancement by Fluosol-DA of melphalan antitumor activity.

The cytotoxicity of melphalan toward exponentially growing FSaIIC fibrosarcoma cells under hypoxia, normal aeration, hyperoxygenation, and stationary phase normally oxygenated cells was examined. Through 4 logs of cell kill by melphalan, there was no difference in survival of FSaIIC cells under any of the four conditions. In the fifth and sixth logs of cell kill, melphalan was most cytotoxic toward normally aerated cells. DNA alkaline elution was performed in FSaIIC cells treated for 1 h with melphalan under the various atmospheres. Both upon immediate elution and after a 6-h delay period the greatest number of DNA cross-links were formed in the normally oxygenated cells. Tumor growth delay studies of the FSaIIC fibrosarcoma treated with melphalan were performed under four levels of oxygenation. From air breathing to 100% oxygen at 3 atm, the tumor growth delay produced by melphalan increased from about 3 days to about 9 days. With the addition of Fluosol-DA, there was an increase in tumor growth delay by melphalan from about 6.5 days with air breathing to about 13 days with 100% oxygen at 3 atm (1 h). When FSaIIC fibrosarcoma tumors were treated with melphalan, and tumor cell survival was measured by colony formation in culture, increasing doses of melphalan produced increasing levels of tumor cell kill in a relatively log linear manner. The addition of Fluosol-DA to treatment with melphalan produced approximately 1 log greater tumor cell kill than melphalan and air breathing under the various oxygenation conditions. There was approximately a 4-fold increase in toxicity to bone marrow granulocyte-macrophage colony-forming units under both extended carbogen breathing conditions (6 h) and hyperbaric oxygenation conditions (100% oxygen, 1 h, 3 atm). The response of the spleen to these various treatment regimens appeared to be immediate and shortlived. Necrotic cells were seen on day 1 posttreatment, with a substantial reduction by day 4 posttreatment. Mitotic figures were essentially absent from the liver on day 1 posttreatment, but by day 4 were significantly increased in treatment groups receiving Fluosol-DA, with the largest number seen in the melphalan/Fluosol-DA with carbogen-breathing group. In conclusion, Fluosol-DA and 1 h of carbogen breathing significantly increases the antitumor activity of melphalan without a concomitant increase in normal tissue toxicity. Although increasing the oxygenation level increased the response of the tumor, normal tissue toxicity was also increased.

Classification of antineoplastic treatments by their differential toxicity toward putative oxygenated and hypoxic tumor subpopulations in vivo in the FSaIIC murine fibrosarcoma.

In order to investigate the effect of environmentally determined conditions on the cytotoxicity of anticancer treatments, Hoechst 33342 dye selected tumor subpopulations were separated after in vivo treatment and plated for single cell colony survival. The 10% brightest cells were assayed as putative normally oxygenated cells and the 20% dimmest as putative hypoxic cells. At single therapeutic doses, cyclophosphamide treatment resulted in the largest differential killing between bright and dim cells (6.3-fold bright greater than dim); 1,3-bis(2-chloroethyl)-1-nitrosourea was 3.2-fold more cytotoxic toward bright cells and carboplatin was 2.4-fold more toxic toward bright cells. Both radiation (10 Gy) and melphalan were 2.2-fold more toxic to bright cells, while cis-diamminedichloroplatinum(II) was 1.8-fold, thiotepa was 1.2-fold and procarbazine was 1.3-fold more toxic to bright cells. Actinomycin D was 3.4-fold more toxic to bright cells. Adriamycin was 2.2-fold, vincristine was 2.1-fold, and etoposide was 1.6-fold more toxic to bright cells. Bleomycin and 5-fluorouracil were also tested and were 1.5- and 2.3-fold more toxic to bright cells, respectively. Only four treatments were more toxic to dim cells: mitomycin C (3.5-fold), misonidazole (1.5-fold), etanidazole (3.5-fold), and 43 degrees C, 30 min local hyperthermia (2.6-fold). In an attempt to shift the pattern of dim cell sparing, Fluosol-DA plus carbogen (95% O2/5% CO2) breathing was added to treatment with radiation (10 Gy), melphalan, cis-diamminedichloroplatinum(II), and etoposide. Although each of these treatments became significantly more toxic with the addition of Fluosol-DA/carbogen, only with melphalan did the combination overcome the sparing of dim cells. These results indicate that cells located distally from the tumor vasculature are significantly less affected by most anticancer drugs and suggest that successful therapeutic strategies against solid tumors will involve greater use of the few treatments which are more toxic toward this tumor subpopulation.

Interaction of hyperthermia and radiation in murine cells: hypoxia and acidosis in vitro, tumor subpopulations in vivo.

To better understand the effect of the level of oxygenation and pH on the heat-radiation interaction, these factors were modeled in vitro using FSaIIC cells in monolayer and correlated with the response of Hoechst 33342 dye-defined FSaIIC tumor subpopulations treated in vivo. Exposure to both 42 degrees C and 43 degrees C for 1 h in culture prior to graded single fractions of radiation resulted in a striking decrease in the radiation oxygen enhancement ratio which was pH as well as temperature dependent. The oxygen enhancement ratio at 37 degrees C and pH 7.40 (or pH 6.45) was 2.9, but decreased to 1.4 at 42 degrees C at normal pH, 1.2 at low pH, and 1.0 at 43 degrees C at both pH values tested. This decrease in the oxygen enhancement ratio resulted from a far more marked decrease in Do values for the radiation survival curves of hypoxic cells compared to normally oxygenated cells at elevated temperatures. In addition, the shoulder region of the radiation survival curves was significantly decreased with increasing temperatures and the magnitude of the decrease was greatest in hypoxic cells at low pH. In vivo treatment followed by immediate tumor excision showed that bright cells (presumably oxygenated cells at normal pH) were approximately 2-fold more sensitive to 10 Gy of radiation than were dim cells (presumably hypoxic cells at low pH) but that dim cells were 2.5-fold more sensitive to 43 degrees C for 30 min hyperthermia. The combination of hyperthermia followed by radiation proved to be 1.8-fold more toxic to dim than to bright cells. Both hyperthermia alone and hyperthermia plus radiation, in contrast to radiation alone, were significantly more cytotoxic when tumors were left in situ for 24 h prior to excision as compared with immediate excision. These results indicate that hyperthermia markedly sensitizes hypoxic cells at low pH to the cytotoxic effects of radiation, as well as effectively killing cells in this tumor subpopulation.