Baseline CD4(+) cell count, not viral load, correlates with virologic suppression induced by potent antiretroviral therapy.

OBJECTIVE: To investigate the relationship between viral load suppression and baseline viral load as well as that between viral load suppression and baseline CD4(+) cell count. DESIGN: Meta-analysis of published and presented studies. METHODS: Trials of two nucleoside analogs plus nevirapine, indinavir, nelfinavir, or efavirenz as therapy for antiretroviral treatment-naive patients with HIV infection or AIDS who were followed-up for at least 6 months were included in the meta-analysis. The proportion of patients with viral loads of <200-500 copies/ml at 6 and 12 months (total number of patients, 1619 and 761, respectively) was regressed to the mean or median baseline viral load and CD4(+) cell count. RESULTS: Thirty-six treatment arms from 30 studies were identified. Multivariate regression demonstrated a significant correlation between baseline CD4(+) cell count and virologic suppression at 6 and 12 months ( t = 2.85, p =.008; and t = 3.08, p =.010, respectively) but not between baseline viral load and virologic suppression ( t = 0.92, p =.365; and t = 1.31, p =.215, respectively). The same pattern was seen in a subanalysis of trials of nevirapine-containing therapy (CD4(+) cell count: t = 2.89, p =.014 at 6 months; viral load suppression: t = 0.84, p =.415). CONCLUSIONS: Baseline CD4(+) cell count was a better predictor of virologic suppression induced by triple combination therapy than was baseline viral load.

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline.

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.

Quantitation of metabolic and radiobiological effects of 6-aminonicotinamide in RIF-1 tumor cells in vitro.

6-Aminonicotinamide (6AN) can be metabolized to 6-amino-NAD(P+), a competitive inhibitor of NAD(P+)-requiring processes, especially the pentose phosphate pathway (PPP) enzyme, 6-phosphogluconate dehydrogenase. The effect of 6AN on the flux of 1 and 6 13C-labeled glucose to lactate, via glycolysis and the PPP, was investigated using 1H-nuclear magnetic resonance. These studies showed that 6AN as a single agent caused a significant 89% (P < 0.0001) inhibition of glycolytic flux but had no detectable effect on the PPP. 31P-nuclear magnetic resonance studies of perifused RIF-1 cells indicated that 4 h of exposure to 6AN were sufficient to cause significant accumulation of 6-phosphogluconate, the substrate for this enzyme (P < 0.0001). A significant reduction in the phosphocreatine: inorganic phosphate ratio was observed under conditions that led to accumulation of 6-phosphogluconate (P < 0.006). Accumulation of 6-phosphogluconate and subsequent reduction in phosphocreatine correlated with significant potentiation of 6 Gy of irradiation by 6AN. These results suggest that the radiation enhancement effect of 6AN may be due to inhibition of glycolysis (mediated by 6-phosphogluconate) and the associated reduction in high-energy phosphates. Additional studies analyzing the metabolic effects of 6AN in combination with radiation are necessary to determine the role of inhibition of the PPP in 6AN enhancement of radiation.

In vivo and ex vivo study of metabolic and cellular effects of 5-fluorouracil chemotherapy in a mouse mammary carcinoma.

The effect of 5-fluorouracil (5FU) on the 31P nuclear magnetic resonance (NMR) profile of a mouse mammary carcinoma, implanted on the foot of CH3/He mice, was studied both in vivo and in perchloric acid extracts. In vivo, significant increases in the ratios, nucleotide triphosphate:inorganic phosphate (Pi) (p < 0.02) and phosphocreatine:Pi (p < 0.005), were observed 48 h after 5FU, relative to control. Two readily resolvable peaks were observed in the phosphomonoester region of the in vivo NMR spectrum, phosphocholine (PC) and a peak (denoted PME') comprised of mainly phosphoethanolamine (PE). PME':PC was significantly elevated relative to control from 24 h to 168 h (p < 0.0001 at 48 h). Perchloric acid extract data indicate that the change in this ratio was due to an increase in the PE concentration rather than a decrease in PC. PE increased from 0.56 +/- 0.11 micromol/g tissue in controls to 0.95 +/- 0.29 micromol/g tissue 48 h after 5FU (p < 0.006). Perchloric acid extracts also revealed a significant increase in phosphodiesters. Glycerophosphocholine increased from 0.82 +/- 0.24 micromol/g tissue in controls to 1.82 +/- 0.61 micromol/g tissue in 5FU treated tumors after 48 h (p < 0.002), and glycerophosphoethanolamine increased from 0.25 +/- 0.06 micromol/g tissue in controls to 0.36 +/- 0.10 micromol/g tissue in treated tumors (p < 0.004). These changes suggest that ethanolamine and choline containing metabolites in this tumor may be metabolized via different pathways. Cell cycle analysis showed only relatively small changes in cell cycle distribution and apoptotic fraction following 5FU.

Effect of radiotherapy and chemotherapy on composition of tumor membrane phospholipids.

Phospholipid extracts were made of a murine mammary adenocarcinoma implanted in the dorsum of the foot of C3H/He mice before and 96 h after 17 Gy irradiation or 150 mg/kg cyclophosphamide. Extracts of untreated tumors, which had grown for a further 96 h, were also studied. Although previous studies have shown significant changes in the precursors and catabolites of phosphatidylcholine and phosphatidylethanolamine following therapy, 31P nuclear magnetic resonance analysis of extracts showed no changes in these membrane constituents and other observed phospholipid species. A significant decrease in 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, however, was observed 96 h following cyclophosphamide treatment.

Effect of 6-aminonicotinamide on the pentose phosphate pathway: 31P NMR and tumor growth delay studies.

6-aminonicotinamide (6AN) has been shown to enhance radiosensitivity in vitro, although previous in vivo studies failed to show an effect. 31P NMR spectra were obtained by using a one-dimensional chemical shift imaging technique on a first generation transplant of the CD8FI spontaneous mammary carcinoma tumor model. Spectra were obtained both before and 10 h after treatment with 6AN (20 mg/kg). Changes in pH, nucleoside triphosphate/inorganic phosphate, and phosphocreatine/ inorganic phosphate measured at 10 h post-6AN were not significant. A new peak was detected 10 h post-6AN, which was assigned to 6-phosphogluconate (6PG), indicating inhibition of the pentose phosphate pathway (PPP). Based on the spectral data demonstrating inhibition of the PPP at 10 h post-6AN, tumor-bearing mice were irradiated (15 Gy x 3 fractions) on Days 1, 10 or 11, and 21 10 h after administration of 6-aminonicotinamide (20 mg/kg). Tumor-bearing mice receiving 6AN alone (20 mg/kg x 3), radiation alone (15 Gy x 3), or saline were also studied. Tumor growth delay studies indicated that 6AN alone induced a small but significant tumor growth delay (4.3 +/- 0.8 days). Radiation alone induced a tumor growth delay of 34.5 +/- 2.7 days. Treatment with 6AN followed by radiation induced a tumor growth delay of 57.0 +/- 3.8 days. This was significantly greater than the TGD values for treatment with 6AN alone or radiation (P < 0.01). No complete regressions were noted after treatment with 6AN or radiation alone. Concomitant therapy with 6AN plus radiation yielded 6/28 complete regressions (21%), which was significantly greater than radiation (P < 0.05) or 6AN alone (P < 0.01) on this mammary carcinoma.

In vivo detection by 31P NMR of pentose phosphate pathway block secondary to biochemical modulation.

The chemotherapeutic regimen of N-(phosphonacetyl)-L-aspartate (PALA) followed 17 h later by 6-methylmercaptopurine riboside (MMPR) and 6-aminonicotanamide (6AN) has been shown to be a potent sensitizer of anti-neoplastic therapy. We undertook this study to compare the therapeutic and metabolic effects of this triple drug combination vs one of its components, 6AN, in a murine mammary carcinoma. After treatment with PALA, MMPR and 6AN, a new peak was detected which was assigned to 6-phosphogluconate (6PG), which is a marker of inhibition of the pentose phosphate pathway at the 6-phosphogluconate dehydrogenase step. Treatment with PALA, MMPR and 6AN also induced a decrease in the ratios of nucleoside triphosphate/inorganic phosphate (NTP/Pi) and phosphocreatine/inorganic phosphate (PCr/Pi) similar to previous results with a different tumor model. These effects were most pronounced at 6 and 10 h. In addition, an increase in PME'/phosphocholine (PME' = downfield peak in the phosphomonoester region) was detected, which was expected because of the cytotoxic effect of this regimen. Treatment with 6AN alone also resulted in the detection of 6PG with a maximum intensity at 6 h post-6AN. Treatment with 6AN alone induced a smaller change in PME'/PC and failed to cause a decrease in PCr/Pi or NTP/Pi at 6 and 10 h. The enhanced response to the combination of PALA, MMPR and 6AN vs 6AN alone, both with regard to cytotoxicity and radiosensitization, may be due to energy depletion.

Study of coumarin metabolism by Chinese hamster lung fibroblasts expressing a human cytochrome P450 using H-nmr.

1. Human cytochrome P450 2A6 is expressed in Chinese hamster lung fibroblasts. The isozyme hydroxylates coumarin at the 7-position. 2. Metabolism of coumarin in these lung fibroblasts was monitored using 1H-nmr. Media samples were taken from cells grown in flasks and also in a fluidized bed bioreactor. In each case 7-hydroxycoumarin was readily observable by nmr in crude extracts of the medium. 3. The rate of formation of 7-hydroxycoumarin observed in the acute bioreactor experiments on a per cell basis was found to be much higher than that obtained under chronic monolayer conditions, and correlated with glucose consumption rates.

13C and 31P NMR investigation of effect of 6-aminonicotinamide on metabolism of RIF-1 tumor cells in vitro.

The effect of 6-aminonicotinamide on the metabolism of RIF-1 tumor cells was investigated using 13C and 31P NMR spectroscopy. 6-Aminonicotinamide can be metabolized to 6-amino-NAD(P), a competitive inhibitor of NAD(P)-requiring processes. 40 microM 6-aminonicotinamide led to an inhibition of 6-phosphogluconate dehydrogenase and an accumulation of 6-phosphogluconate. A subsequent accumulation of the 6-phosphogluconate precursor 6-phosphoglucono-delta-lactone was observed in the 13C NMR spectrum. These metabolites were shown to be intracellular, although a small amount of leakage of 6-phosphoglucono-delta-lactone occurred. The intracellular concentrations of 6-phosphogluconate and 6-phosphoglucono-delta-lactone were 1.9 +/- 0.8 micromol/108 cells (+/-1 standard deviation) and 0.8 +/- 0.4 micromol/10(8) cells, respectively, after 15 h. Glucose utilization and lactate production were significantly inhibited by 6-aminonicotinamide (both p < 0.05), indicating inhibition of glycolysis. 31P NMR data showed that phosphocreatine was significantly depleted in cells exposed to 6-aminonicotinamide (p < 0.05). Exposure of RIF-1 cells to 6-aminonicotinamide prior to 3- or 6-Gy x-irradiation induced a supra-additive cell kill, indicating that 6-aminonicotinamide is acting as a radiosensitizer. There was no effect of 6-aminonicotinamide alone or when the drug was given postradiation, suggesting that its mechanism of action may be by inhibition of radiation-induced repair.

In vivo and in vitro studies of cyclophosphamide chemotherapy in a mouse mammary carcinoma by 31P NMR spectroscopy.

The effect of cyclophosphamide on the metabolic profile of a mammary carcinoma implanted on the foot of mouse was studied by 31P NMR spectroscopy both in vivo and in perchloric acid extracts. The ratio nucleotide triphosphate:P(i) was significantly elevated in cyclophosphamide treated tumours relative to untreated tumours after 96 h in vivo (p < 0.05). Phosphocreatine:P(i) was similarly elevated from 48 to 168 h (p < 0.01). Resolution of the phosphomonoester peak into two distinct resonances allowed us to estimate the ratio of PME' to phosphocholine (PC), where PME' is a composite peak consisting, in part, of phosphoethanolamine (PE). PME':PC was found to be significantly higher in treated animals relative to control animals in vivo (p < 0.01 from 48 to 168 h). Perchloric acid extract spectra suggest that the increase in PME':PC was in part due to a decrease in PC concentration and also due to an increase in a previously unidentified resonance which was coresonant with PE. Extract data show that there was a significant increase in the concentration of the phosphodiesters, glycerophosphocholine (p < 0.01) and glycerophosphoethanolamine (p < 0.05) in treated relative to control tumours. The changes in the phosphomonoester resonances are qualitatively similar to previously described changes following radiation and suggest that they may be a marker of cell kill or lack of cell growth after antineoplastic therapy.

A 1H/15N n.m.r. study of nitrogen metabolism in cultured mammalian cells.

1. Heteronuclear 1H/15N n.m.r. experiments are described in which 15N labelling of cellular metabolites is detected via their proton resonances. 2. These n.m.r. experiments have been used to monitor label redistribution amongst extracellular metabolites in cultures of mammalian cells incubated with L-[2-15N]glutamine, L-[5-15N]glutamine and 15NH4Cl. Label redistribution was monitored in two HeLa cell lines and in two CHO cell lines which showed a range of extractable activities of glutamate dehydrogenase, glutaminase and glutamine synthetase. 3. In cells incubated with L-[2-15N]glutamine the 15N label was subsequently found in a number of metabolites including alanine, aspartate, glycine and pyrrolidone-5-carboxylic acid. There was no detectable production of 15NH4+, showing that most of the glutamate formed in the reaction catalysed by glutaminase was subsequently transaminated rather than oxidatively deaminated by glutamate dehydrogenase. 4. Incubation of cells with L-[5-15N]glutamine showed that the ammonia in the cultures was derived predominantly from the amide group of glutamine. 5. The rate of formation of L-[5-15N]glutamine in cells incubated with 15NH4Cl was used to estimate glutamine synthetase flux in vivo. Flux in this reaction was only observable in the two CHO cell lines which express relatively high levels of the enzyme.

Contact sensitivity to captafol in BALB/c mice.

The fungicide captafol has been reported as causing irritant and allergic contact dermatitis in humans and in guinea pigs. This study investigated the ability of purified captafol to cause contact sensitization in BALB/c mice. Female mice were pretreated with an intraperitoneal injection of cyclophosphamide or saline. Applications of captafol (18.7 or 37.4 mg/ml), dinitrofluorobenzene (DNFB; 5 mg/ml) or solvent (4:1 acetone:ethanol mixture) were administered to the shaved abdomen on 2 consecutive days, or on days 1, 2, 8, 15, 22 and 29. Following challenge with captafol (37.4 mg/ml) or DNFB (2 mg/ml) on the right ear 6 days after the last induction, ear thickness ratios (right ear/left ear) were significantly larger after challenge in captafol-induced and DNFB-induced mice compared to control mice. A slightly larger response was observed with the smaller induction dose level of captafol and with multiple inductions over the course of a month. The overall maximum response to captafol was not increased by pretreatment with cyclophosphamide. Histologically, ears from captafol-induced and DNFB-induced mice showed edema and cellular infiltration. This study demonstrated the ability of captafol to produce contact hypersensitivity in the BALB/c mouse.

Diet and colon cancer: assessment of risk by fiber type and food source.

How fiber in the diet is related to the development of colon cancer was assessed in a population-based study conducted on 231 cases and 391 controls in Utah between 1979 and 1983. Crude fiber consistently decreased risk associated with colon cancer in both males [odds ratio (OR) = 0.4] and females (OR = 0.5). Dietary fiber, as analyzed by the method of A. S. Bitner, and neutral detergent fiber were not consistently related to colon cancer risk. Of the noncellulose polysaccharides examined, mannose and galactose were protective against cancers in the ascending colon in males (ORs = 0.5 and 0.3, respectively), whereas galactose and uronic acid were protective against cancers in the ascending colon in females (ORs = 0.5). Highest quartiles of intake of fruits and vegetables were also associated with a decreased risk of colon cancer in males (ORs = 0.3 and 0.6, respectively) and in females (ORs = 0.6 and 0.3, respectively) compared with lowest quartile of intake, whereas high intake of grains was not protective.

Unique purified hydrated-gelatin diet for feeding dietary fiber to Wistar rats.

A purified hydrated gelatin diet was developed for feeding dietary fibers to Wistar rats. A dry fiber mix was prepared that consisted of 54.91 dextrose, 13.80 casein, 2.97 AIN mineral mix, 1.28 AIN vitamin mix, 0.17 dl-methionine, 6.80 lard and 5.10 gelatin (g/100g total dry feed). Either cellulose, hemicellulose, lignin, or pectin (15 g/100 g total dry feed) was added to the hydrated dry fiber mix and blended until complete distribution and hydration of the fiber was achieved. After gelling, these hydrated diets were stable for up to 24 hours in environmental conditions commonly encountered in animal facilities. Gel weep was minimal thus permitting feed consumption to be monitored conveniently by weighing the residue in the feeders. In situ examination of stomach contents after feeding such hydrated diets to rats indicated that the gelatin gel was readily degraded and did not confound gel formation by fiber itself. Feed efficiency values (g gain/100 kcal digestible energy) for these diets following a 26-day feeding trial were as follows: no fiber, 6.21; cellulose, 6.38; hemicellulose, 6.23; lignin, 6.52; and pectin, 5.53.

Allergic hypersensitivity to the insecticide malathion in BALB/c mice.

The widely used insecticide malathion (diethylmercaptosuccinate, S ester with O,O-dimethylphosphorodithioate) has been reported to cause allergic responses in some exposed people and in guinea pigs. In this study, IgE antibody-mediated and cell-mediated hypersensitivity to malathion was evaluated in female BALB/c mice. To elicit malathion-specific antibodies of the IgE class, a conjugate of the anhydride of the diacid metabolite of malathion (MMA) with keyhole limpet hemocyanin was administered ip with aluminum hydroxide as adjuvant. Sera collected following three sequential sensitizations were tested for specific IgE with the passive cutaneous anaphylaxis (PCA) test in Sprague-Dawley rats. MMA coupled to bovine serum albumin was used as the challenge antigen. Specific IgE was produced following the second and third sensitization in the mice receiving 10 and 100 micrograms of conjugate and following the third sensitization in the mice receiving 1 microgram of conjugate. A paper radioallergosorbent test (PRAST) was as sensitive as and showed a good correlation with the PCA assay for samples analyzed by both procedures. Malathion applied epicutaneously on 2 days or over 4 weeks failed to elicit delayed-type hypersensitivity as determined by change in ear thickness, 5-[125I]iodo-2'-deoxyuridine incorporation in the ear, and histology of the ear following ear challenge, with or without pretreatment of the mice with cyclophosphamide. MMA-specific IgE antibodies were not detected by the PCA test in the serum of mice treated epicutaneously for 4 weeks.

Allergic hypersensitivity to the herbicide 2,4-D in BALB/c mice.

Following several reports of human allergic hypersensitivity, the ability of 2,4-D (2,4-dichlorophenoxyacetic acid) to elicit 2,4-D-specific IgE antibodies and delayed-type hypersensitivity in BALB/c mice was studied. 2,4-D-specific IgE antibodies were detected in mouse sera following the second intraperitoneal immunization with 1, 10, or 100 micrograms 2,4-D-keyhold limpet hemocyanin conjugate with aluminum hydroxide adjuvant. Specific IgE was determined with the rat passive cutaneous anaphylaxis test using a conjugate of 2,4-D with bovine serum albumin for challenge. The highest antibody titers and a measurable response in all mice were seen in the group that received 1 microgram of 2,4-D conjugate. Dinitrophenyl-specific IgE was measured at all intervals examined in mice immunized with a dinitrophenyl-keyhole limpet hemocyanin conjugate. 2,4-D applied epicutaneously on 2 d or over 4 wk failed to elicit delayed-type hypersensitivity as measured by change in ear thickness, incorporation of 5-[125I]iodo-2'-deoxyuridine, or histology following challenge on the ear. No 2,4-D-specific IgE antibodies were detected in serum during the 4-wk sensitization period. Similar treatment with a known sensitizer, dinitrofluorobenzene, produced delayed hypersensitivity. Following 4 wk of dosing, low titers of dinitrophenyl-specific IgE antibodies were elicited.

Accumulation and depletion of cadmium and lead in tissues and milk of lactating cows fed small amounts of these metals.

Groups of lactating cows were fed 0, 40, and 200 mg of added cadmium (as chloride) and 0, 100, and 500 mg of added lead (as acetate) per animal per day in separate experiments. Milk and blood were sampled periodically and analyzed for concentrations of metal ions. Metal feeding was discontinued after 3 mo, and selected animals were necropsied for tissue residue studies. Remaining animals were continued on control ration for another 3 mo and then killed and tissues obtained. Cadmium feeding did not produce a dose-related increase of this metal in blood, milk, or skeletal muscle. Liver and kidneys were the primary organs of cadmium accumulation, and concentration of cadmium in these organs continued to rise during 3 mo of feeding the control diet after the initial exposure period. Lead did not accumulate in skeletal muscle but showed a dose-related increase in blood, milk, bone, liver, and kidney. In most tissues there was a rapid decline of lead concentrations after cessation of treatment, except in bone. Low dietary intake of cadmium and lead do not produce an appreciable rise of these metals in edible products, e.g., milk or meat. Of the tissues analyzed, liver and kidney accumulate both cadmium and lead, and cadmium especially persists in these organs for long periods. Bone is the primary site of deposition for lead but not cadmium.

Applicator exposure to 2,4-D, dicamba, and a dicamba isomer.

Potential respiratory and dermal exposure to applicators were estimated in a ground boom spray application of 2,4-D and dicamba. Time-weighted averages for airborne herbicide residues did not exceed 2.2 microgram/cu.m. in the cabs of application vehicles allowing only minor respiratory exposure. Dermal exposure was important as relatively large amounts of 2,4-D (1.2 - 18 mg) and dicamba (0.32-6.6 mg) were rinsed from applicators' hands. Urine analysis showed that the maximum elimination of herbicides occurred between 16 and 40 h after terminating exposure. A dicamba isomer (20% of the active material in the commercial formulation) was excreted in higher concentrations than dicamba in applicators' urine suggesting different toxicokinetic properties for the two compounds.

The need for comprehensive diet studies to assess the relation of lipids to cancer.

Dietary lipids have been linked by both basic research and epidemiological evidence to the etiology of some cancers. It is yet unclear which lipid(s) may be active in the carcinogenic process, but one promising hypothesis concerns the interaction of cholesterol metabolites, considered a risk factor for colon cancer, and dietary fiber which may have a protective role. A multidisciplinary case control study currently underway is investigating the relationship and possible mode of action of fiber and bile acids on colon cancer. The study has epidemiological, biochemical, and pharmacological components that have been designed to integrate data on the intake and fate of lipids, dietary fiber, and other nutritional parameters in colon cancer cases and matched controls and in animal models. Subcomponents of the study deal specifically with the characterization of dietary fiber constituents and their in vivo effect on lipid metabolism.