Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Response to adrenocorticotropic hormone or corticotrophin-releasing hormone and vasopressin in lactating cows fed an immunomodulatory supplement under thermoneutral or acute heat stress conditions.

Adrenal responsiveness was tested in nonpregnant, lactating Holstein dairy cows fed diets supplemented with OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ), an immune modulator, and in nonsupplemented control (CON) cows following bolus infusions of a combination of corticotropin-releasing hormone (CRH; 0.3 µg/kg of BW) and arginine vasopressin (VP; 1.0 µg/kg of BW) or ACTH (0.1 IU/kg of BW) in 2 environments: thermoneutral [TN; temperature-humidity index (THI) <60] for 24 h/d and heat stress (HS; THI >68 for 17 h/d). Cows (506) were initially fed OG (n = 254) or CON (n = 252) diets for 44 d before selection of a subgroup of cows (n = 12; 6 OG, 6 CON) for the study. The 2 subgroups were balanced for parity, milk yield, and days in milk. All cows were transported to and housed in 2 environmentally controlled rooms at the University of Arizona Agricultural Research Complex (Tucson). Cows were given 3 d to acclimate to the rooms and then underwent 12 d of TN conditions and then 8 d of HS conditions for a total of 24 d on experiment. Cows were infused with CRH-VP on d 9 of TN and on d 1 of HS and with ACTH on d 10 of TN and on d 2 of HS. Hormone infusions took place at 1000 h (0 h) on each infusion day. Blood samples, taken in 30-min intervals, were first collected at 0800 h (-2 h) and were drawn until 1800 h (8 h). Before infusion, serum progesterone was elevated in OG cows compared with CON cows. Infusion of releasing factors (CRH-VP or ACTH) caused increases in serum cortisol and progesterone, but cortisol release was greater in CON cows than in OG cows during HS, whereas progesterone did not differ between the 2 treatments. Serum ACTH increased following infusion of releasing factors, but this increase was greater following CRH-VP infusion than ACTH infusion. Serum bovine corticosteroid-binding globulin also increased following infusion of releasing factors in both treatment groups, but this increase was greater during HS in cows fed OG. The free cortisol index (FCI) increased following CRH-VP and ACTH and was higher in HS than in TN for both OG and CON cows. However, the FCI response was blunted in OG cows compared with CON cows during HS. Heat stress enhanced the adrenal response to releasing factors. Additionally, the adrenal cortisol and FCI response to releasing factors was reduced during acute heat stress in cows fed OG. Collectively, these data suggest that OG supplementation reduced the adrenal responsiveness to factors regulating cortisol secretion during acute HS.

Short communication: Blood mineral and gas concentrations of calves born to cows fed prepartum diets differing in dietary cation-anion difference and calcium concentration.

Eighty-two multiparous Holstein cows were fed diets differing in dietary cation-anion difference (DCAD) and Ca concentrations in a randomized block design experiment beginning 4 wk before anticipated calving to determine the effects on colostrum yield and quality and acid-base balance and mineral status of newborn calves. Treatments were arranged as a 2 × 2 factorial to provide 2 DCAD [-22 mEq/100 g of dry matter (NEG) or -3 mEq/100 g of dry matter (NEU)] and 2 supplemental Ca concentrations (1.3 or 1.8% of dry matter). After calving, cows were milked within 2 to 8 h and colostrum yield was recorded. Calves were fed 200 g of IgG of a commercial colostrum replacer within 4 h of birth. No differences were observed in birth weight or dystocia score among treatments, which averaged 42.7 kg and 1.12, respectively. Colostrum yield was not different among treatments and averaged 8.75 kg. Colostrum quality, as measured using a Brix refractometer, was not affected by DCAD but was higher for 1.3% compared with 1.8% Ca: 21.58% and 19.87%, respectively. Colostrum IgG concentrations were higher for NEG compared with NEU and for 1.3% compared with 1.8% Ca. No differences were observed in concentrations of serum IgG, Ca, P, K, Cl, anion gap, or whole-blood pH, partial pressure of O2, or SO2 of calves among treatments. Serum Mg and lactate concentrations were higher and CO2 tended to be lower for calves born to cows fed 1.3% compared with 1.8% Ca. Interactions of DCAD and Ca were observed for serum Na and Cl, which were higher for NEU-1.3% Ca and NEG-1.8% Ca compared with NEU-1.8% Ca and NEG-1.3% Ca. Whole-blood partial pressure of CO2, and HCO3 exhibited an interaction of DCAD and Ca and tended to be lower for NEU-1.3% Ca and NEG-1.8% Ca compared with NEU-1.8% Ca and NEG-1.3% Ca. Results of this trial indicate that feeding prepartum diets with 1.8% compared with 1.3% supplemental Ca reduced colostrum quality and serum concentrations of Mg and lactate in calves immediately after birth. Feeding NEG supported higher colostrum IgG concentrations. Blood mineral concentrations and blood gas balance tended to differ, but the effects were not consistent across DCAD and Ca.

A commercial immune modulating feed additive restores L-selectin and CCL5 expression following dexamethasone treatment of murine immune cells in a MyD88-dependent manner.

Bovine mastitis costs the dairy industry billions of dollars every year and presents a health challenge in dairy facilities. Immunosuppressive effects of the periparturient period increase the incidence of mastitis. During this time, cattle experience an elevation in circulating cortisol, which reduces polymorphonuclear cell function and ability to clear infection. OmniGen-AF (OMN; Phibro Animal Health, Teaneck, NJ) is an immunomodulatory feed additive that alters gene expression and is used to reduce rates of mastitis. We hypothesized that OMN restores gene expression during periods of immune stress through inhibiting the suppressive effects of glucocorticoid receptor signaling on Toll-like receptor signaling. To test our hypothesis, wild-type (WT) or MyD88 knockout mice were supplemented with OMN and challenged with lipopolysaccharide following dexamethasone (Dex) treatment. Polymorphonuclear cell and macrophage RNA was isolated from intraperitoneal lavages and analyzed for gene expression profiles. Treatment of mice with Dex suppressed expression of l-selectin and CCL5 as compared with phosphate-buffered saline treatment of WT mice. Expression of l-selectin and CCL5 was significantly reduced with Dex treatment in control-fed but not OMN-supplemented WT mice. The protective effect of OMN supplementation on l-selectin expression during Dex treatment was abolished in MyD88 knockout mice. These results suggest that OMN supplementation restores responses of certain genes suppressed by Dex in immune cells in a MyD88-dependent manner. Future research will determine the specific Toll-like receptors, transcription factors, and biochemical properties of OMN that restore gene expression in immunosuppressed cells.

Can the two mechanisms of tumor cell killing by radiation be exploited for therapeutic gain?

The radiation killing of tumor cells by ionizing radiation is best described by the linear-quadratic (LQ) model. Research into the underlying mechanisms of α- and ß-inactivation has suggested that different molecular targets (DNA in different forms) and different microdosimetric energy deposits (spurs versus electron track-ends) are involved. Clinical protocols with fractionated doses of about 2.0 Gy/day were defined empirically, and we now know that they produce cancer cures mainly by the α-inactivation mechanism. Radiobiology studies indicate that α and ß mechanisms exhibit widely different characteristics that should be addressed upfront as clinical fractionation schemes are altered. As radiation treatments attempt to exploit the advantages of larger dose fractions over shorter treatment times, the LQ model can be used to predict iso-effective tumor cell killing and possibly iso-effective normal tissue complications. Linking best estimates of radiobiology and tumor biology parameters with tumor control probability (TCP) and normal tissue complication probability (NTCP) models will enable us to improve and optimize cancer treatment protocols, delivering no more fractions than are strictly necessary for a high therapeutic ratio.

A comparison of susceptibility to photodynamic treatment between endothelial and tumor cells in vitro and in vivo.

BACKGROUND: Photodynamic therapy (PDT) is being widely used for treatment of cancer and non-malignant diseases. The mechanisms of phototoxicity to solid tumor are not yet completely understood. Knowledge of the inherent sensitivity of endothelial cells in comparison to tumor cells would be helpful to predict tumor response to PDT, and thereby optimize treatment protocols. METHODS AND RESULTS: The intrinsic sensitivity of rodent endothelial and tumor cells to PDT was studied using an in vitro clonogenicity assay that strictly controlled for light and photosensitizer exposure, as well as cellular photosensitizer and oxygen concentration. Taking into consideration cell size, ploidy and glutathione content, no significant difference in sensitivity to phototoxicity was observed between tumor and endothelial cells. Electron microscopy studies were also conducted to examine endothelial and tumor cells for differential cellular damage following interstitial PDT of rat prostate tumor. No evidence for selective damage to endothelial cells was demonstrated. CONCLUSIONS: Rodent tumor cells and endothelial cells are equally susceptible to Photofrin-mediated PDT damage. Sufficient photosensitizer accumulated in solid tumor seems to be one of the key factors for PDT effectiveness.

Radiosensitization of tumour cells by cantharidin and some analogues.

PURPOSE: Mammalian cells at mitosis contain chromatin in compacted form and are hypersensitive to ionizing radiation. Previous research had shown some chemicals that induce chromatin compaction within interphase cells act as radiosensitizers. Of these agents, cantharidin (LS-1), which is an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), showed good radiosensitizing activity at non-toxic doses. Cantharidin and 13 additional structural analogues (LS-2-14) were tested for their radiosensitizing activity on tumour cells in vitro. MATERIALS AND METHODS: Twelve of the 14 cantharidin analogues were synthesized in the authors' laboratory. Various concentrations of the drugs were screened for toxicity and radiosensitizing effectiveness with asynchronous DU-145 (human prostate carcinoma) cells. More detailed radiobiological studies of the more potent agents were performed with HT-29 (human colon carcinoma) cells since they could be readily synchronized. The radiosensitization of G1 phase HT-29 cells was measured after a 2-h exposure to the more potent drugs and reductions of the surviving fraction after an acute dose of 2 Gy (SF2Gy) served to estimate their relative effectiveness. The increase in phosphorylation of histone 1 (H1) and histone 3 (H3) induced by these drug exposures was measured by Western blotting of protein extracts. Drug-induced change in chromatin morphology was visualized by electron microscopy, and the alkaline comet assay (which measures DNA single-strand breaks) was employed to measure the radiation sensitivity of cellular chromatin in the drug-treated cells. RESULTS: Of the 14 cantharidin analogues tested, LS-1, LS-2 and LS-5 at concentrations of 3-20 microM showed little or no toxicity, produced elevated levels of H1 and H3 phosphorylation, and effected significant radiosensitization at low radiation dose. The chromatin in tumour cells treated with LS-5 became visibly compacted and its DNA was about 1.6 times more sensitive to radiation-induced strand breakage relative to that of control cells. CONCLUSIONS: The results confirm the authors' earlier studies that showed an increase in tumour cell intrinsic radiosensitivity by exposure to agents that promote chromatin compaction. LS-5 was identified as the optimal radiosensitizing agent of this class of compounds. Radiosensitization was correlated with chromatin compaction and elevated phosphorylation of H1 and H3. The DNA in drug-treated cells exhibited an enhanced sensitivity to radiation-induced single-strand breakage.

Single-hit mechanism of tumour cell killing by radiation.

PURPOSE: To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. MATERIALS AND METHODS: Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. RESULTS: The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. CONCLUSIONS: Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

The radiation hypersensitivity of cells at mitosis.

PURPOSE: Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. MATERIALS AND METHODS: Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. RESULTS: Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. CONCLUSIONS: The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Chemical agents that promote chromatin compaction radiosensitize tumour cells.

PURPOSE: Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential. MATERIALS AND METHODS: Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy. RESULTS: OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms. CONCLUSIONS: These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

Hypoxia in human prostate carcinoma: an Eppendorf PO2 study.

The purpose of this study was to characterize the extent of hypoxia in human prostate carcinoma using the Eppendorf PO2 microelectrode. Custom-made Eppendorf PO2 microelectrodes were used to obtain PO2 measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies), as well as from a region of normal muscle for comparison. Fifty-nine patients with localized prostate cancer were studied, all of whom received brachytherapy implants under spinal anesthesia. A multivariate mixed effects analysis for prediction of tumor oxygenation was performed including the following covariates: type of tissue (prostate versus muscle), prostatic-specific antigen, disease stage, patient age and race, tumor grade, volume, perineural invasion, and hormonal therapy. Because of differences in patient characteristics, control measurements were obtained from normal muscle in all patients. This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median PO2 = 2.4 mm Hg) compared with the measurements from normal muscle (average median PO2 = 30.0 mm Hg), p < 0.0001. A multivariate, linear, mixed analysis demonstrated that the only significant predictor of oxygenation was the type of tissue (prostate versus muscle). This study, using in vivo electrode oxygen measurements, suggests that hypoxia exists in human prostate carcinoma. More patients will be accrued to this study to ultimately correlate the oxygenation status in prostate carcinoma tumors with treatment outcome.

Chromatin compaction and tumor cell radiosensitivity at 2 gray.

Mammalian cells at mitosis, differentiated lymphocytes, and some radiation-hypersensitive mutants in interphase contain all or a measurable portion of their chromatin in condensed/compacted form and are hypersensitive to ionizing radiation by the mechanism described by single-hit inactivation kinetics (alpha). These observations led to the investigation as to whether compacted chromatin in interphase is the target that determines the widely variable alpha-parameters and surviving fractions of 2 Gy (SF2Gy) measured for human tumor cell lines. Six cell lines whose SF2Gy ranged from 0.29 to 0.73 were used for this study. Their different radiosensitivities were associated mainly with differences in their single-hit inactivation parameters (alpha). Electron microscope images of interphase nuclei were optically scanned, and the pixel densities were digitized for quantitative analyses. A significant correlation between the percentage of nuclear pixels with densities similar to those found in mitotic chromosomes (percent compacted chromatin) and the alpha-inactivation parameters was observed. Digital analyses of electron and/or confocal microscope images of chromatin in interphase tumor cells in biopsy specimens could become a rapid assay for predicting the intrinsic radiosensitivity of tumor clonogens. This research has also identified some inhibitors of protein (histone) phosphatases that promote chromatin compaction and radiosensitize cells to 2-Gy dose fractions.

Increased hypoxia correlates with increased expression of the angiogenesis marker vascular endothelial growth factor in human prostate cancer.

OBJECTIVES: To test the hypothesis that increasing levels of hypoxia are associated with increased expression of vascular endothelial growth factor (VEGF) in prostate cancer by correlating the level of median tissue oxygenation in human prostate tumors with the immunohistochemically determined level of VEGF expression. METHODS: Custom-made Eppendorf oxygen microelectrodes were used to quantitate the pO(2) levels in prostate tumors of 13 men undergoing radical prostatectomy. All pO(2) measurements were performed under fluorine-based general anesthesia. Paraffin-embedded tumor tissue from these men was analyzed to measure the level of VEGF expression by immunohistochemical staining. The significance of the associations between the pO(2) levels and VEGF staining were determined by the Pearson correlations. RESULTS: The range of the median pO(2) levels (based on between 97 and 129 individual measurements) among 13 prostate tumors was 0.5 to 44.9 mm Hg. The blinded comparison of pO(2) levels and VEGF staining intensity demonstrated a significant correlation between increasing hypoxia and the percentage of cells staining positive for VEGF (r = -0.721, P = 0.005). This correlation was also significant when pO(2) levels were compared with the overall immunoreactive score, which takes into account staining intensity (r = -0.642, P = 0.018). CONCLUSIONS: To our knowledge, this is the first study demonstrating a significant association between increasing levels of hypoxia and increased expression of the angiogenesis marker VEGF in human prostate carcinoma. The results of our study further support the exploration of antiangiogenesis strategies for the treatment of human prostate cancer.

Marking hypoxia in rat prostate carcinomas with beta-D-[125I]azomycin galactopyranoside and.

UNLABELLED: The purpose of this study was to determine, with a rodent tumor model, if microelectrode measurements of unmodulated tumor oxygenation predict for the avidity of hypoxic markers to tumor tissue. METHODS: The rapidly growing, anaplastic variant of the Dunning rat prostate carcinoma cell line (R3327-AT) was implanted subcutaneously on the upper backs of Fischer X Copenhagen rats. Approximately 100 measurements of PO2 were obtained from tumors of 5-10 g in animals that were restrained and then subjected to different anesthetic procedures. Values of median PO2 (in mm Hg) and percentage of measurements <5 mm Hg obtained from individual tumors were used to define tumor oxygenation status. The radiodiagnostic hypoxic markers beta-D-iodinated azomycin galactopyranoside (IAZGP) and [99mTc]HL-91 were simultaneously administered to 26 animals whose tumor oxygen levels had been measured. Six hours after marker administration, the animals were killed; tumor, blood, and muscle tissues were sampled; and percentage injected dose per gram (%ID/g*), tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) parameters were determined. Parameters of marker avidity to individual tumors were linearly correlated with microelectrode measurements of tumor oxygenation to determine the significance of inverse associations. RESULTS: The median PO2 of 41 tumors varied from 2.0 to 20.9 mm Hg, with an average value of 7.5 +/- 1.4 mm Hg. Six tumors had unusually high values; that is, >10 mm Hg, and when these were excluded from the analysis, the average median PO2 of the remaining 35 was 4.3 +/- 0.7 mm Hg. When electrode measurements of tumor oxygenation were obtained under conditions of halothane anesthesia with the animals breathing O2, carbogen, or air, median PO2 values increased significantly (P = 0.001). When animals were deeply anesthetized by intraperitoneal injection of ketamine-xylazine, median PO2 values were not significantly different (P = 0.13) from those obtained while the animals were restrained and breathing air. There was no inverse correlation of significance between the electrode measurements of median PO2 and the avidity of beta-D-IAZGP nor [99mTc]HL-91 in this tumor model. The range of median PO2 values in these tumors was at least 3 mm Hg, and the range of hypoxic marker avidity was less than twofold. CONCLUSION: These data demonstrate that microelectrode measurements of rat tumor oxygenation did not correlate with the avidity of the two hypoxic markers, at least in this tumor model. The larger dynamic range of tumor oxygen measurements obtained with microelectrodes might be biased to low values by their necrotic fractions, the zones within solid tumors that contain dead cells and debris that will not be labeled by bioreducible hypoxic markers. Hypoxic marker avidity to individual tumors will have to be validated by other assays that can predict for their radiosensitivity.

Single-photon emission computed tomography and positron-emission tomography assays for tissue oxygenation.

Radiotherapy prescription can now be customized to target the major mechanism(s) of resistance of individual tumors. In that regard, functional imaging techniques should be exploited to identify the dominant mechanism(s). Tumor biology research has identified several mechanisms of tumor resistance that may be unique to radiation treatments. These fall into 3 broad areas associated with (1) tumor hypoxic fraction, (2) tumor growth rate, (3) and the intrinsic radiosensitivity of tumor clonogens. Imaging research has markers in various stages of development for quantifying relevant information about each of these mechanisms, and those that measure tumor oxygenation and predict for radioresistance are the most advanced. Positron-emission tomography (PET) measurement of oxygen 15 has yielded important information, particularly about brain tissue perfusion, metabolism, and function. Indirect markers of tumor hypoxia have exploited the covalent binding of bioreductive intermediates of azomycin-containing compounds whose uptakes are inversely proportional to intracellular oxygen concentrations. Pilot clinical studies with single-photon emission computed tomography (SPECT) and PET detection of radiolabeled markers to tumor hypoxia have been reported. Recently, other studies have attempted to exploit the reduction properties of both technetium and copper chelates for the selective deposition of radioactive metals in hypoxic tissues. A growing number of potentially useful isotopes are now available for labeling several novel chemicals that could have the appropriate specificity and sensitivity. Preclinical studies with "microSPECT" and "microPET" will be important to define the optimal radiodiagnostic(s) for measuring tissue oxygenation and for determining the time after their administration for optimal hypoxic signal acquisition. Radiolabeled markers of growth kinetics and intrinsic radiosensitivity of cells in solid tumors are also being developed. We conclude that radiation oncology is uniquely positioned to benefit from functional imaging markers that identify important mechanisms of tumor radioresistance, since several strategies for overcoming these individual mechanisms have already been identified.

Increasing levels of hypoxia in prostate carcinoma correlate significantly with increasing clinical stage and patient age: an Eppendorf pO(2) study.

BACKGROUND: The purpose of this study was to analyze the extent of hypoxia in prostate carcinoma tumors using the Eppendorf pO(2) microelectrode and correlate this with pretreatment characteristics and prognostic factors. METHODS: Custom-made Eppendorf pO(2) microelectrodes were used to obtain pO(2) measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies) as well as from a region of normal muscle for comparison. Each set of measurements comprised approximately 100 separate readings of pO(2), for a total of 10,804 individual measurements. Fifty-five patients with localized prostate carcinoma were studied: Forty-one patients received brachytherapy implants, and 14 patients underwent radical prostatectomy. The pO(2) measurements were obtained in the operating room by using a sterile technique under spinal anesthesia for the brachytherapy group and under general anesthesia for the surgery group. The Eppendorf histograms were recorded and described by the median pO(2), mean pO(2), and percentage < 5 mm Hg and < 10 mm Hg. A multivariate mixed-effects analysis for the prediction of tumor oxygenation was performed and included the following covariates: type of tissue (prostate vs. muscle), type of treatment (implant vs. surgery) and/or anesthesia (spinal vs. general), prostate specific antigen level, disease stage, patient age and race, tumor grade, tumor volume, perineural invasion, and hormonal therapy. RESULTS: Due to differences in patient characteristics and the anesthesia employed, control measurements were obtained from normal muscle (in all but two patients). This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median pO(2), 9.9 mm Hg) compared with the measurements normal muscle (average median pO(2), 28.6 mm Hg; P < 0.0001). A multivariate, linear, mixed analysis demonstrated that, among all of the patients, the significant predictors of oxygenation were tissue (prostate vs. muscle) and anesthesia (spinal vs. general) or treatment (implant vs. surgery). Among the brachytherapy (spinal anesthesia) patients, the significant predictors of pO(2) were tissue type, disease stage, and patient age. There were no significant predictors of oxygenation in the surgical (general anesthesia) group. CONCLUSIONS: This study, employing in vivo electrode oxygen measurements, demonstrated that hypoxia exists in prostate carcinoma tumors. A dramatic effect of anesthesia was observed, likely due to modulation of polarography in the presence of fluorine. Within the group of brachytherapy (spinal anesthesia) patients, increasing levels of hypoxia (within prostatic tissue) correlated significantly with increasing clinical stage and patient age. More patients will be accrued to this prospective study to further correlate the oxygenation status in prostate carcinoma tumors with known prognostic factors and, ultimately, treatment outcome.