Tissue pH Values Research Articles

Modulation of G-protein activation, calcium currents and opioid receptor phosphorylation by the pH-dependent antinociceptive agonist NFEPP.

N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide is a newly-designed pain killer selectively activating G-protein-coupled mu-opioid receptors (MOR) in acidic injured tissues, and therefore devoid of central side effects which are typically elicited at normal pH values in healthy tissues. However, the neuronal mechanisms underlying NFEPP's antinociceptive effects were not examined in detail so far. Voltage-dependent Ca2+ channels (VDCCs) in nociceptive neurons play a major role in the generation and inhibition of pain. In this study, we focused on the effects of NFEPP on calcium currents in rat dorsal root ganglion (DRG) neurons. The inhibitory role of the G-protein subunits Gi/o and Gβγ on VDCCs was investigated using the blockers pertussis toxin and gallein, respectively. GTPγS binding, calcium signals and MOR phosphorylation were also investigated. All experiments were performed at acidic and normal pH values using NFEPP in comparison to the conventional opioid agonist fentanyl. At low pH, NFEPP produced more efficient G-protein activation in transfected HEK293 cells and significantly reduced VDCCs in depolarized DRG neurons. The latter effect was mediated by Gβγ subunits, and NFEPP-mediated MOR phosphorylation was pH-dependent. Fentanyl's responses were not affected by pH changes. Our data indicate that NFEPP-induced MOR signaling is more effective at low pH and that the inhibition of calcium channels in DRG neurons underlies NFEPP's antinociceptive actions.

A New Ratio-Metric pH Probe, “ThiAKS Green” for Live-Cell pH Measurements

Deviation of the H+ concentration from optimum values within the organelles is closely associated with irregular cellular functions that cause the onset of various diseases. Therefore, determining subcellular pH values in live cells and tissues is valuable for diagnostic purposes. In this study, we report a novel ratiometric fluorescence probe 1H-pyrazole-3-carboxylic acid, 4-(benzo[d]thiazol-2-yl)-3-(2,4-dihydroxy-3-methylphenyl)-1H-pyrazole-5-carboxylicacid4-(2-benz othiazolyl)-5-(2,4-dihydroxy-3-methylphenyl), to which we will refer as ThiAKS Green (Thiazole AKyol shifting green), that is pH sensitive. The results presented here show that the probe can penetrate the cell membrane in less than 30 minutes and does not show any detectable toxicity. The measured color shifts up on pH change are linear and most significant around physiological pH (pKa=7.45), thus making this probe suitable for live-cell imaging and intracellular pH measurements. During the long-incubation periods following the application of the probe and the fluorescent microscopy measurements, it shows stable properties and is easy to detect in live cells. In conclusion, the results suggest that ThiAKS Green can be used to obtain precise information on the H+ distribution at various compartments of the live cells.

Establishment of incontinence-associated dermatitis rat models and assessment of the therapeutic effects of zinc oxide, painless skin protective film and silicone dressing.

The aim of the present study was to construct incontinence-associated dermatitis (IAD) rat models and observe the therapeutic effects of zinc oxide, painless skin protective film and silicone dressing on IAD. A total of 54 rats were randomly divided into nine groups: i) Control group; ii) trypsin model group; iii) model + zinc oxide group; iv) model + painless skin protective film group; v) model + silicon dressing group; vi) synthetic urine combined with trypsin model group (joint model group); vii) joint model + zinc oxide group; viii) joint model + painless skin protective film group; and ix) joint model + silicone dressing group. A total of 4 days after applying the zinc oxide, protective film or silicon dressing intervention, IAD scores and pH values in skin tissues were examined. Skin tissues and blood samples were collected. Hematoxylin and eosin staining, immunohistochemical staining of major histocompatibility complex class II (MHC-II) and western blot analysis of MHC-II, NF-κB/p65, phosphorylated (p)-NF-κB/p65, STAT1 and p-STAT1 were carried out in skin tissue. Serum IFN-γ, IL-1β, IL-2 and TNF-α levels were determined using ELISA. The results demonstrated that IAD scores and pH values were both higher in the model groups than the control, which were significantly ameliorated by silicone dressing. The skin tissue structure of IAD rats both in trypsin model group and joint model group was severely damaged, the wounds were not covered by epidermis, and numerous inflammatory cell infiltrations were observed. After treatment, dermatitis was improved. Skin tissue from the trypsin and joint IAD models had higher MHC-II, NF-κB p65, p-NF-κB p65, STAT1 and p-STAT1 expression than controls, which was decreased by protective film and silicon dressing. Zinc oxide reduced NF-κB p65, p-NF-κB p65, STAT1 and p-STAT1 expression. However, no significant differences were observed in NF-κB/p-NF-κB ratio and STAT1/p-STAT1 ratio among groups. Furthermore, serum IFN-γ, IL-1β, IL-2 and TNF-α levels were significantly elevated in trypsin and joint IAD rats. The upregulation of these cytokines was significantly inhibited after all three treatments. Among the three treatment methods, silicone dressing had the best therapeutic effect. Thus, these findings revealed that zinc oxide, painless skin protective film and silicone dressing could ameliorate the severity of IAD rat models, and that silicone dressing possessed the best therapeutic effect.

Development of Neutral pH-Responsive Microgels by Tuning Cross-Linking Conditions.

Polymer microgels that respond in a range of neutral pH can be useful for the development of molecular imaging tools and drug-delivery carriers. Here, we describe a simple approach in developing microgels that undergo volume phase transitions and substantial nuclear magnetic resonance (NMR) relaxometric changes within a narrow pH range of 6.4 to 7.4. The pH-responsive microgels were synthesized using methacrylic acid and a series of ethylene glycol dimethacrylate cross-linkers with repeating units of ethylene glycol that range from one to four. NMR relaxometry demonstrated that the transverse relaxation time (T2) of a suspension containing microgels that were cross-linked with diethylene glycol dimethacrylate sharply decreases at the pH where volume phase transition occurs. The polymer microgels cross-linked with 40 and 45 mol% of diethylene glycol dimethacrylate caused about 50% T2 reduction with decreasing pH from 6.8 to 6.4. These results demonstrated that responses of microgels to a range of neutral pH can be easily tuned by using appropriate cross-linkers with certain cross-linking degree. This approach can be useful in developing highly sensitive molecular sensors for magnetic resonance imaging (MRI) of tissue pH values.

Polymers with tertiary amine groups for drug delivery and bioimaging

Over the past decade, biopolymers have gained great interests especially in biomedicine due to their physical properties and/or chemical structures changes in response to external stimuli in a certain time frame or at a specific location. Among them, poly( β -amino ester)s, methacrylate-based block copolymers and polypeptide with tertiary amine groups have been extensively studied and exhibit pH sensitive properties due to the protonation of tertiary amine groups. The pH values in normal organs, tissues, and subcellular compartments are always different from those in pathological tissues. These interesting properties allow their applications in a variety of fields ranging from diagnosis and therapeutics of diseases. Here, we review the recent progress of poly( β -amino ester)s, methacrylate-based block copolymers and polypeptide with tertiary amine groups and their applications in drug delivery and bioimaging.

A ratiometric two-photon probe for quantitative imaging of mitochondrial pH values.

Mitochondrial pH (pHmito) is known to be alkaline (near 8.0) and has emerged as a potential factor for mitochondrial function and disorder. We have developed a ratiometric two-photon probe (CMP1) for quantitative analysis of pHmito in live cells and tissues. This probe is designed to function by controlling the intramolecular charge transfer from 2-naphthol, having an ideal pKa value (7.86 ± 0.05) in the cells to monitor pHmito. This transition results in a marked yellow to red emission color change in response to pH alterations from 6.0 to 9.0. CMP1 exhibits easy loading, selective and robust staining ability of mitochondria, low cytotoxicity, and bright two-photon excited fluorescence in situ, thereby allowing quantitative imaging of the pHmito in live cells and tissues. The ratiometric TPM imaging clearly reveals that subcellular distribution of the pHmito values is heterogeneous, with the pHmito values in the perinuclear region being higher than those at the periphery of the cells. The changes of pHmito values on carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and autophagic processes were also investigated along with their morphological alterations at specific subcellular positions. We also used CMP1 to visualize the pHmito values of Parkinson's disease model astrocytes as well as living hippocampal tissues. Our results demonstrate that CMP1 will be useful as a quantitative imaging probe to study pHmito in biomedical research.

PH imaging of mouse kidneys in vivo using a frequency-dependent paraCEST agent.

This study explored the feasibility of using a pH responsive paramagnetic chemical exchange saturation transfer (paraCEST) agent to image the pH gradient in kidneys of healthy mice. CEST signals were acquired on an Agilent 9.4 Tesla small animal MRI system using a steady-state gradient echo pulse sequence after a bolus injection of agent. The magnetic field inhomogeneity across each kidney was corrected using the WASSR method and pH maps were calculated by measuring the frequency of water exchange signal arising from the agent. Dynamic CEST studies demonstrated that the agent was readily detectable in kidneys only between 4 to 12 min postinjection. The CEST images showed a higher signal intensity in the pelvis and calyx regions and lower signal intensity in the medulla and cortex regions. The pH maps reflected tissue pH values spanning from 6.0 to 7.5 in kidneys of healthy mice. This study demonstrated that pH maps of the kidney can be imaged in vivo by measuring the pH-dependent chemical shift of a single water exchange CEST peak without prior knowledge of the agent concentration in vivo. The results demonstrate the potential of using a simple frequency-dependent paraCEST agent for mapping tissue pH in vivo. Magn Reson Med 75:2432-2441, 2016. © 2015 Wiley Periodicals, Inc.

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D (1)H and (31)P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. (1)H-(1)H homonuclear and (1)H-(31)P heteronuclear correlation experiments enabled the direct assessment of the (1)H-(31)P spin systems for signals that suffered from overlapping in the 1D (1)H spectra, and linked the information present in the 1D (1)H and (31)P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the (31)P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from (31)P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH(3))(3) signals of phosphocholine and choline in (1)H spectra of the tissue in these tumour samples.

Measurement of pH Values in Human Tissues by Two‐Photon Microscopy

pH values go live! A ratiometric two-photon (TP) probe (NP1, see scheme) that has a significant TP action cross-section, high photostability, negligible toxicity, and can estimate pH values in live cells and human tissues by two-photon microscopy is described. NP1 can detect the difference in pH between live cells from the gastroesophageal junction (GEJ) and the lower esophageal sphincter of patients with and without esophagitis.

Measurement of pH Values in Human Tissues by Two‐Photon Microscopy

Sichtbare pH-Werte: Die ratiometrische Zweiphotonensonde NP1 mit signifikantem Wirkungsquerschnitt ist hoch lichtbeständig und kaum toxisch. Durch Zweiphotonenmikroskopie mit ihr können ungefähre pH-Werte in lebenden Zellen und menschlichem Gewebe ermittelt werden. NP1 erkennt pH-Unterschiede zwischen lebenden Zellen des Ösophagus-Magen-Übergangs und des unteren Ösophagus-Schließmuskels von Ösophagitis-Patienten und gesunden Probanden (siehe Bild).

Agaricus bisporus as a source of tyrosinase for phenol detection for future biosensor development

Phenols are toxic compounds that are present in several industrial wastewaters, so their quantification has great environmental importance. In order to permit an analytical methodology for in situ monitoring, this work aims to study the application of Agaricus bisporus tissue as a source of tyrosinase and the optimum reaction conditions for the development of a phenol biosensor. Such an enzyme is a polyphenol oxidase that transforms many different phenolic compounds into quinones. Experiments with fungi tissue were performed to evaluate different sizes of tissue (0.5, 1.0 and 1.5 cm), different temperatures (23.5°C to 60°C), and different pH values (6, 7 and 8) to quantify analytically phenol content. Amongst the tested conditions, those that had presented larger efficiency in phenol oxidation were attained with the fungal tissue size of 1 cm, at pH 8.0, in the temperature range from 35°C to 45°C.

Response of cytoplasmic pH to anoxia in plant tissues with altered activities of fermentation enzymes: application of methyl phosphonate as an NMR pH probe.

Acidification of the cytoplasm is a commonly observed response to oxygen deprivation in plant tissues that are intolerant of anoxia. The response was monitored in plant tissues with altered levels of lactate dehydrogenase (LDH) and pyruvate decarboxylase (PDC) with the aim of assessing the contribution of the targeted enzymes to cytoplasmic pH (pH(cyt)) regulation. The pH(cyt) was measured by in vivo (31)P nuclear magnetic resonance (NMR) spectroscopy using methyl phosphonate (MeP) as a pH probe. The potential toxicity of MeP was investigated by analysing its effect on the metabolism of radiolabelled glucose. MeP accumulated to detectable levels in the cytoplasm and vacuole of plant tissues exposed to millimolar concentrations of MeP, and the pH-dependent (31)P NMR signals provided a convenient method for measuring pH(cyt) values in tissues with poorly defined signals from the cytoplasmic inorganic phosphate pool. Pretreatment of potato (Solanum tuberosum) tuber slices with 5 mm MeP for 24 h did not affect the metabolism of [U-(14)C]glucose or the pattern of (14)CO(2) release from specifically labelled [(14)C]-substrates. Time-courses of pH(cyt) measured before, during and after an anoxic episode in potato tuber tissues with reduced activities of LDH, or in tobacco (Nicotiana tabacum) leaves with increased activities of PDC, were indistinguishable from their respective controls. MeP can be used as a low toxicity (31)P NMR probe for measuring intracellular pH values in plant tissues with altered levels of fermentation enzymes. The measurements on transgenic tobacco leaves suggest that the changes in pH(cyt) during an anoxic episode are not dominated by fermentation processes; while the pH changes in the potato tuber tissue with reduced LDH activity show that the affected isozymes do not influence the anoxic pH response.

Nitrite protonation as a necessary stage in the generation of nitric oxide from nitrite in biological systems

The yields of nitric oxide from 1 mM and 10 mM sodium dithionite in 5 or 150 mM solutions of HEPES buffer (pH 7.4) differed by a factor of 200. Dithionite acted as both a strong reducing agent and an agent responsible for local acidification of the solutions without significant changes in pH. The concentration of nitric oxide was estimated by electron paramagnetic resonance (EPR) by monitoring its incorporation into water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which resulted in the formation of EPR-detectable mononitrosyl complexes of iron. Ten seconds after dithionite addition, the concentration of mononitrosyl iron complexes reached 2 μM, whereas it did not become greater than 0.01 μM in 5 mM HEPES buffer. It has been suggested that this difference results from a longer lifetime of a localized decrease in pH in a weaker buffer solution. This time could be long enough for the protonation of some nitrite molecules. Nitrous acid thus formed decomposed to nitric oxide. A difference in nitric oxide formation from nitrite in weak and strong buffer solutions was also observed in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, in the weak buffer the nitric oxide yield was only three-four times greater than in the strong buffer. An increase in the nitric oxide yield from nitrite was observed in solutions containing both proteins. A significant amount of nitric oxide from nitrite was formed in mouse liver preparation subjected to freezing and thawing procedure followed by slurrying in 150 mM HEPES buffer (pH 7.4) and dithionite addition (10 mM). We suggest that the presence of zones with lowered pH values in cells and tissues may be responsible for the predominance of the acidic mechanism of nitric oxide formation from nitrite. The contribution of nitric oxide formation from nitrite catalyzed by heme-containing proteins as nitrite reductases may be minor under these conditions.

Changes in tissue pH and temperature after incision indicate acidosis may contribute to postoperative pain.

Incisional pain is a common form of acute pain. Previously, the authors studied persistent pain behaviors caused by incisions, using animal models for postoperative pain. In this study, the authors measured tissue pH and hind paw temperature before and after incision to understand factors that may activate and sensitize nociceptors in the incision. Rats underwent a plantar incision, a gastrocnemius muscle incision, or a cutaneous paraspinal incision. For the hind paw incision, pain behaviors were measured. Tissue pH was measured using a pH-sensitive needle electrode in halothane-anesthetized rats. The pH in the incision was compared to a corresponding control site on the contralateral side of the rat or to the sham-operated group. Plantar tissue pH was 7.16 +/- 0.04 in sham-operated rats. Ten minutes after plantar incision, tissue pH was decreased to 6.91 +/- 0.20 (P < 0.05), and this decrease was sustained through 60 min after incision, when pH was 6.99 +/- 0.06 (P < 0.05). Tissue pH values were 6.95, 6.90, 6.89, and 6.95 (P < 0.05 vs. sham) 4 h and 1, 2, and 4 days after incision, respectively. On postoperative day 7, when plantar pH was same as for the control side (7.13 +/- 0.05), guarding behavior, heat responses, and responses to mechanical stimuli recovered. Outside the incised area in the hind paw, tissue pH was normal. Tissue pH was significantly correlated with all pain behaviors. In the gastrocnemius muscle, tissue pH was 7.14 +/- 0.7 in the sham-operated side. Ten minutes after incision, tissue pH was 6.54 +/- 0.12 (P < 0.05), and muscle pH remained decreased through 60 min after gastrocnemius incision when pH was 6.76 +/- 0.17 (P < 0.05). Tissue pH was also significantly decreased (P < 0.05) on day 1 (6.96 vs. 7.20) and day 4 (7.06 vs. 7.18) after gastrocnemius incision but was not reduced on postoperative day 8 (7.11 vs. 7.15). A paraspinal incision also decreased tissue pH in the hairy skin of the rat compared with the preincision value. Hind paw skin temperature did not change after incision. A decrease in pH occurs immediately after incision and is sustained for at least 4 days. During the period of decreased tissue pH, pain behaviors are evident. When the tissue pH returns to normal, pain behaviors are diminished. The decreased pH is localized at the incision site and not to areas surrounding the incision. Decreased pH likely contributes to nociceptor sensitization and pain related behaviors after incision. The magnitude of the pH change varies among tissues. An increase in hind paw skin temperature does not play a role in these pain-related behaviors.

The Effect of Tissue pH Values of a Wound on Pain Behaviors in an Acute Pain Model of the Rat

Background: There is a suggestion that tissue acidosis may contribute to the induction of pain in inflammation and ischemia. High local proton concentrations have been found in painful inflammatory and ischemic conditions. We assessed tissue pH and pain behavior in a rat model for postoperative pain. Methods: An incision of skin, fascia, and muscle was performed in the right foot. Pain behaviors to von Frey, blunt probe stimulation, and resting pain were evaluated on the incised feet. Then tissue pH was measured in each of the five rats at 4 hours, 1, 2, 4, and 7 days after incision. Results: The withdrawal threshold to von Frey was decreased, the response frequency to blunt probe and pain scores were elevated from 4 hours to 1-2 days after the incision. The deep tissue pH decreased from pH 7.16 in sham to pH 6.95, 6.90, 6.89, and 6.95 at 4 h and 1, 2, and 4 days after incision respectively. Conclusions: In this study, the tissue pH of the wound decreased after surgery. However, the change of tissue pH was not always consistent with pain behaviors. Our results suggest that tissue acidosis may relate partially to the induction of pain after tissue injury.

PH regulation in acid-stressed leaves of pea plants grown in the presence of nitrate or ammonium salts: studies involving 31P-NMR spectroscopy and chlorophyll fluorescence

31P nuclear magnetic resonance spectroscopy was used to monitor changes of cytoplasmic and vacuolar pH values in leaf tissues from young pea plants which had been grown in hydroponic culture with either nitrate or ammonium salts as sources of nitrogen. When acid stress was applied by the addition of 15% CO 2 to air (5.1 mM CO 2 in solution), cytoplasmic pH values decreased fast by 0.5 pH units and then increased slowly without reaching the initial pH, while vacuolar pH values decreased by 0.1 pH units. Under anaerobic conditions, the cytoplasmic pH decreased by one pH unit and the vacuolar pH increased by almost 0.4 pH units. These changes were rapidly reversed when CO 2 was removed from air or, after anaerobiosis, by aeration. However, with mannose present during and after anaerobiosis, aeration failed to bring pH values back to the levels observed before anaerobiosis. Simultaneously, mannose phosphates accumulated and cytoplasmic phosphate disappeared. Since loss of phosphate decreases ATP levels, the observations suggest that ATP-dependent pumping of protons into the vacuole restored the cytoplasmic pH partially during acidification by CO 2 and fully after anaerobiosis. Photosynthesis was initially inhibited by high CO 2 and then restored indicating that protons are exported not only across the tonoplast into the vacuole but also across the chloroplast envelope into the cytosol. No large differences in pH regulation were observed in leaves of pea plants which were grown with either nitrate or ammonium salts. Apparently, retarded growth of ammonium-fertilized plants cannot be attributed to ineffective pH regulation.

Physiological implications of hyperbaric oxygen tensions in isolated limb perfusion using melphalan: a pilot study.

Controversy exists concerning the optimal pO2 of the perfusate during isolated limb perfusion (ILP) with melphalan. Therefore we studied the implications of hyperbaric oxygen tensions in the perfusate. In 12 consecutive patients, subcutaneous pO2 (Continucath 1000), tissue and tumor pH, and blood gas values were monitored throughout the ILP procedure. ILP started with an oxygen flow through the bubble oxygenator which was set routinely at one half of the flow of the perfusate; 30 min before the end of ILP, the oxygen flow was tripled. Mean arterial pO2 before and during ILP (before and after increasing the oxygen supply) was 19.4, 25.5 and 49.4 kPa, respectively. Mean subcutaneous pO2 values before, during (before and after increasing the oxygen supply), and post-ILP, were 7.4, 10.1, 16.3, and 9.1 kPa, respectively. Tissue pH values in the subcutis and muscle decreased during routine oxygen supply (p = 0.001); muscle pH moved towards starting values after increase of the oxygen supply (p = 0.011). In 4 patients, tumor pH was recorded showing a rise after increasing the oxygen supply (from 7.10 to 7.22; p = 0.11). In conclusion, high pO2 in the perfusate improves muscle pH during ILP. However, a concomitant rise in tumor pH may unfavorably influence the therapeutic effect of ILP, as it has been shown that low pH increases the cytotoxicity of melphalan.

Tissue pH in cold-stored human donor livers preserved in University of Wisconsin solution. A noninavasive clinical study with 31P-magnetic resonance spectroscopy.

It is not known whether the tissue acidosis that accompanies cold storage is the beginning of irreversible cell injury, ultimately leading to cell death, or whether it is a natural "protective" mechanism for cells to survive hypoxic periods. To answer this question, the tissue pH of 45 cold-stored human donor livers preserved in University of Wisconsin solution (UW) was assessed shortly before implantation using noninvasive 31P magnetic resonance spectroscopy. We conclude that tissue pH during cold storage may be partly dependent upon hepatic glycogen stores and donor age. The wide range of tissue pH values that was observed at the time of implantation does not result in significant effects on cellular damage after transplantation. This indicates that tissue pH is not a major determinant for the viability of UW solution-preserved human donor livers, as indicated by postoperative hepatocellular damage and liver synthesis function. The membrane stabilizing and buffering capacity of UW solution appears to protect liver viability against tissue acidosis. Our results also indicate that liver tissue pH can be lower than has been previously assumed in the literature without significant adverse effects on liver viability.

Physiologically based pharmacokinetics of methoxyacetic acid: dose-effect considerations in C57BL/6 mice.

Methoxyacetic acid (MAA), a weak acid with a pKa of 3.57, was used to test the broad hypothesis that distribution of weak acids in maternal and fetal tissues is determined principally by the pKa of the acid and the pH values of tissue and fluid compartments and to examine tissue dose-teratogenesis relationships, as well as administered dose-teratogenesis relationships. Five related experimental studies were conducted in pregnant C57BL/6CrIBR mice: a conventional dose-response study of developmental toxicity and transplacental pharmacokinetics in mice, a second dose-response study in which reproductive outcomes in litters from individual dams were related to individual pharmacokinetic behavior, a protein-binding experiment, an embryo tissue localization study, and determination of pH in maternal and embryonic compartments after exposure to MAA. MAA was administered intraperitoneally at 9:00 a.m. on day 10 of gestation, at doses ranging from 88 to 164 mg/kg. Localization within the forelimb bud of the embryo, an MAA target site, was determined by computerized image analysis of the distribution of radiolabeled MAA. The kinetic predictions of a physiologically based model incorporating tissue pH values and MAA pKa agreed well with observed concentrations at the lowest dose. However, at intermediate and higher doses, concentrations in both maternal and embryonic tissues were consistently underestimated. MAA was bound neither to maternal plasma proteins nor to embryonic proteins. Intermediate and higher doses of MAA caused dose-dependent transient depressions in tissue pH, but these were not of sufficient duration to bring predicted tissue concentrations into congruence with the concentrations observed. Distribution of MAA within the forelimb bud was broadly consistent with the pH hypothesis, but MAA concentration was not increased in the distal postaxial sector that is the site of the precursor cells of the missing digits. Internal exposure to MAA, defined as the area under the maternal plasma or embryo concentration curve (AUC), was not proportional to administered dose, but AUC-response relationships generated by the group and individual dose-response studies were comparable. While AUC may be a useful measure of effective MAA dose, it cannot be accurately predicted at teratogenic doses of this agent by the model as it is presently structured.

Microregional distributions of glucose, lactate, ATP and tissue pH in experimental tumours upon local hyperthermia and/or hyperglycaemia.

Microregional distributions of glucose, lactate and ATP concentrations as well as tissue pH values were determined in subcutaneous rat tumours during normothermia and normoglycaemia, and upon local hyperthermia (HT) and/or hyperglycaemia (HG). Experiments were performed in order to investigate whether, and to what extent, these adjuvant therapeutic measures applied alone or in combination can modify the bioenergetic and metabolic status, parameters that are known to markedly influence the therapeutic response of tumours to heat. Local HT was performed in a saline bath (44 degrees C/2 h) and HG was induced by i.v. infusion of glucose for 2.5 h (blood glucose levels during heating: 35-40 mM). Immediately after treatment, the microregional distributions of glucose, lactate and ATP concentrations were assessed using quantitative bioluminescence and single-photon counting. In corresponding histological sections the fraction of tumour tissue with changes indicating cellular damage was determined. For comparison, global levels of glucose, lactate, ATP, ADP and AMP were measured using enzymatic assays or HPLC. Tumour tissue pH values were recorded immediately after treatment with miniaturised needle glass pH electrodes. Upon HT alone, the microregional glucose distribution remained unchanged. Lactate concentrations significantly increased, resulting in a pH drop of about 0.20 pH units. Mean ATP concentrations decreased without an obvious change in the shape of the distribution curve. The fraction of tumour tissue showing cellular damage increased from 18% (in control tumours) to 27%. Upon HG alone, mean glucose and lactate levels in the tumours increased. Glucose, lactate and pH distributions became broader. Lactate accumulation results in a severe tumour acidosis (mean pH = 6.22). Mean ATP concentrations marginally decreased despite a higher glucose availability, probably because of poorer ATP yield resulting from changes in metabolic channelling (Crabtree effect). The fraction of tumour tissue exhibiting cellular damage was 23%. Following the combined treatment (HT/HG), glucose and lactate levels, and tissue pH were similar to those seen upon HG alone. However, ATP concentrations were lowest under this condition. The variation of tumour ATP concentrations is substantially reduced with only a few tumour areas remaining with ATP levels of at least 0.6 mumol/g. The ATP depletion upon HT/HG is accompanied by a drastic increase in the fraction of tissue areas exhibiting cellular damage to 61%. It may therefore be concluded that only the combined treatment can deplete ATP to such an extent that a pronounced cytotoxic effect is achieved.