pH frequency distributions of tumours grown s.c. from 30 human tumour xenograft lines in rnu/rnu rats were analysed with the use of H+ ion-sensitive semi-microelectrodes prior to and following stimulation of tumour cell glycolysis by i.v. infusion of glucose. At normoglycemia, the average pH of the tumours investigated was 6.83 (range, 6.72-7.01; n = 268). Without exception, all xenografts responded to the temporary increase in plasma glucose concentration (PGC) from 6 +/- 1 to 30 +/- 3 mM by an accumulation of acidic metabolites, as indicated by a pH reduction to an average value of 6.43 (range, 6.12-6.78; n = 292). This pH value corresponds to a ten-fold increase in H+ ion activity in tumour tissue as compared to arterial blood. Tumour pH approached minimum values at 2-4 h after the onset of glucose administration and could be maintained at acidic levels for 24 h by controlled glucose infusion. Irrespective of pH variations between tumours grown from individual xenograft lines, there was no major difference in pH response to glucose between the four main histopathological tumour entities investigated, i.e. breast, lung and gastrointestinal carcinomas, and sarcomas. In tumours from several xenograft lines, an increase in blood glucose to only 2.5-times the normal value (14 mM) was sufficient to reduce the mean pH to 6.4. Glucose-induced acidosis was tumour-specific. The pH frequency distributions in liver, kidney and skeletal muscle of tumour-bearing rnu/rnu rats were only marginally sensitive to hyperglycemia (average pH, 6.97 vs normal value of 7.14). Tumour-selective activation of pH-sensitive anti-cancer agents, e.g. alkylating drugs, acid-labile prodrugs or pH-sensitive immunoconjugates may thus be feasible in a wide variety of human cancers.

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The urinary excretion of the DNA alkylation products 3-methyladenine (3-MeAde) and 3-ethyladenine (3-EtAde) after controlled exposure to cigarette smoke over a period of 4 days was determined by competitive radioimmunoassay after separation by HPLC. Twenty-four hour urine samples were collected from five smokers and five non-smokers. Days 1 and 3 (control days) were without smoking, on days 2 and 4 smokers consumed 24 cigarettes each within 8 h in an unventilated room (45 m3) in the presence of non-smokers. Average levels of carbon monoxide during exposure were 15-20 p.p.m., 2.8-3.5 mg/m3 of respirable suspended particles and 75-86 micrograms/m3 of nicotine. Carboxyhemoglobin levels increased by 9.0 and 1.8% in smokers and passive smokers respectively. On control days, urinary excretion of 3-MeAde was similar in smokers and non-smokers (4.7-6.2 micrograms/24 h). Smoking resulted in a significant increase (P < 0.01) in 3-MeAde excretion (13.6-14.8 micrograms/24 h); no change was observed in the average excretion of 3-MeAde by passive smokers (4.8-4.9 micrograms of 3-MeAde/24h). Baseline 3-EtAde excretion on control days was similar in smokers and passive smokers (13.7-32.8 ng/24 h). In smokers, the amount of urinary 3-EtAde was increased > 5-fold (119.3-138.5 ng/24 h) on smoking days; no effect on 3-EtAde excretion was observed on average in passive smokers (18.0-25.2 ng/24 h). The nature of the DNA-reactive agent(s) responsible for the increased urinary excretion of 3-alkyladenines, in particular of the sensitive indicator 3-EtAde, remains to be determined.

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK(a) = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm(2)), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2-5.3 in all root zones. Addition of 1 mM NH(4)(+) caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO(3)(-) was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO(3)(-), NO(3)(-) elicited a higher and persistent alkalization (0.06-0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H(+)-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO(3)(-) provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging.

The relationship between the apoplastic pH in young sunflower roots (Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe 21 into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fe III reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe 21 complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe III reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe III reduction; ammonium had the reverse effect. Nitrate together with HCO 3 -, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe III reduction in the apoplast, and hence Fe 21 uptake into the cytosol.