Chronic nephropathy in ad libitum overfed Sprague-Dawley rats and its early attenuation by increasing degrees of dietary (caloric) restriction to control growth.

The early development and progression of chronic nephropathy and its amelioration by moderate and marked dietary restriction (DR) was determined in Sprague-Dawley (SD) rats at 20, 33, 60, and 113 weeks of age. Both sexes of SD rats were overfed ad libitum (AL) or DR-fed at 72-79%, 68-72%, or 47-48% of the adult AL intake. The AL-fed rats rapidly developed increased body and kidney size, increased glomerular area (GA) and urinary protein loss, followed by declining creatinine clearance. Early increased kidney growth and glomerular hypertrophy by 20 weeks preceded increases in glomerular sclerotic index (GSI), 7-day BrdU tubular labeling index (TLI), and the lesions associated with chronic nephropathy. The glomerular number (GN) or the number of nephrons did not differ between the groups over the course of the study. Moderate DR (68-79% of AL) prevented the increased kidney size and GA at 20 weeks and delayed increases in GSI and TLI until 60 weeks of age. Marked DR (47-48% of AL) prevented increases in kidney size, GA and TLI at 20 weeks, and GSI at 60 weeks of age. In AL-fed rats, the early increase in GA predicted the early onset of proteinuria and the later decrease in creatinine clearance, and increased GSI, TLI, and mortality from severe nephropathy. The temporal and dose-related effects of increasing degrees of DR demonstrated that while nephron numbers were unchanged with age, the early development of glomerular hypertrophy was the critical morphological biomarker predicting the progression and severity of chronic nephropathy. Caloric restriction by DR prevented or delayed the development of glomerulosclerosis, tubulointerstitial damage, functional changes, morbidity, and mortality associated with chronic nephropathy in AL-overfed SD rats by controlling initial body and kidney growth, glomerular size, and nephron hypertrophy. These results indicate that control of body and renal growth by DR may be essential to prevent the development and progression of glomerulosclerosis in spontaneous nephropathy of laboratory rats.

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis.

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

Magnetic resonance spectroscopy and imaging methods for measuring tumour and tissue oxygenation.

It is well known that low levels of tissue oxygen (pO2) protect tumour cells from ionising radiation and some chemotherapeutic agents. Thus, numerous studies have been aimed at developing methods to measure tissue oxygenation. An initial discussion of some of the traditional methods for measuring oxygenation is included, followed by a discussion of magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) methods for measuring tumour and normal tissue oxygenation. The latter methods are of interest because of the non-invasive nature of magnetic resonance (MR). Some of the MR methods described herein include: 31P MRS, 1H MRS and MRI, and 19F MRS and MRI. Each method is detailed, including a brief assessment of its ability to measure tumour oxygenation and its potential for clinical application.

The effect of blood flow modification on intra- and extracellular pH measured by 31P magnetic resonance spectroscopy in murine tumours.

Intra- and extracellular pH (pHi and pHe) were measured simultaneously by 31P magnetic resonance spectroscopy (MRS) in CaNT tumours before and after blood flow modification. Before modification, pHi was 7.1 +/- 0.09 (n = 11) and pHe [measured with an MRS-visible extracellular marker, 3-aminopropyl phosphonate (3-APP)] was 6.7 +/- 0.05 (n = 8). Chemical shift imaging and localised MRS experiments showed that the 3-APP signal was only from the tumour, not surrounding tissue. After modification by vascular occlusion, independent of whether tumours were maintained at room temperature (22-24 degrees C) or kept warm (33-35 degrees C), there was a decrease in pHi and pHe with pHi decreasing to a greater extent. Qualitatively similar results were found using flavone acetic acid (FAA) as a blood flow modifier; only four out of nine tumours responded to FAA. Concomitant with the reduction of the pH gradient after modification was a decrease in the phosphorylation state of the adenine nucleotides measured either as ATP/Pi by MRS or [ATP]/[ADP][P(i)] in tumour extracts. These results indicate that the intracellular uptake of chemotherapeutic drugs which are dependent on the transmembrane pH gradient will not be enhanced in cells made ischaemic as a result of vascular shutdown.

Effect of vasoactive drugs on tumour blood flow as determined by 2H nuclear magnetic resonance spectroscopy.

A selective reduction in tumour blood flow (TBF) could enhance the effects of hyperthermia treatment and of drugs toxic to hypoxic cells. Vasodilator-induced changes in TBF were monitored in transplanted rat fibrosarcomas by non-invasively measuring the uptake of D2O using 2H nuclear magnetic resonance spectroscopy. Hydralazine (1 or 5 mg kg-1) caused a large (45%) reduction in mean arterial blood pressure (MABP) and a 40-60% reduction in TBF. Low-dose hydralazine (0.1 mg kg-1) caused a 20% reduction in MABP but no significant change in TBF. The doses of prazosin (1 mg kg-1) and calcitonin-gene related peptide (CGRP, 1 nmol kg-1) which caused a 20% reduction in MABP led to a 50-60% reduction in TBF. These results demonstrate the advantage of prazosin and CGRP over hydralazine for the reduction of TBF despite a small hypotensive effect. CGRP may be the most suitable of these agents for clinical use because of its short physiological half-life.

A method for direct in vivo measurement of drug concentrations from a single 2H NMR spectrum.

The use of 2H-labeled drugs provides a measure of drug concentration in situ directly from a single 2H NMR spectrum obtained with any antenna by correcting only for differential saturation effects. The limit of detection for a drug labeled with three equivalent deuterons is roughly 0.5 mM.

Flavone acetic acid (NSC 347512)-induced modulation of murine tumor physiology monitored by in vivo nuclear magnetic resonance spectroscopy.

Flavone acetic acid (FAA), a new drug with broad activity against transplanted solid tumors of mice, induces nonrepairable DNA single strand breaks that correlate with therapeutic efficacy. To test the hypothesis that the inability of the cells to repair single strand breaks is associated with a disruption of tumor energy metabolism, in vivo 31P nuclear magnetic resonance (NMR) spectra were acquired from s.c. implanted Glasgow osteogenic sarcomas in C57BL/6 x DBA/2 F1 mice both before and after treatment with FAA i.v. at 100, 150, or 200 mg/kg and from a control (no treatment) group (n = 4 in each group). While FAA produced a dose-dependent decrease in both the nucleoside triphosphates level and pH, only treatment with an efficacious dose of 200 mg/kg resulted in both a reduction in pH and a complete loss of nucleoside triphosphates from the NMR spectrum at 4 h with no recovery until 48 h and little recovery out to 72 h. The ATP concentration determined by high pressure liquid chromatography in a parallel set of experiments was 5.59 +/- 1.16 (SE) mumol/g (wet weight) in control tumors (n = 9) and 0.24 +/- 0.12 mumol/g (wet weight) at 4 h after 200 mg/kg FAA (n = 7). To examine the possibility that the loss of ATP and decreased pH are associated with a reduction in tumor blood flow, we used 2H NMR to monitor the washout of D2O injected directly into the tumor both before and 4 h after treatment with 200 mg/kg FAA. The pretreatment tumor blood flow of 12.4 +/- 1.7 ml/min/100 g was reduced to 1.9 +/- 0.5 ml/min/100 g at 4 h after treatment (n = 3). The FAA-induced reduction of both tumor blood flow and ATP may play an important role in its mechanism of action and should be considered in the combination of FAA with other drugs or therapeutic modalities. In addition, because 31P NMR can be used clinically, it should provide a nonambiguous early indicator of activity for clinical trials of FAA.

The development of children's strategies for the social control of emotion.

It has been shown that young children can recognize emotional states in their peers, that they understand many of the antecedents of emotion, and they are motivated to change negative emotional states in others. The present study examined children's ability to nominate strategic social action that would alter the ongoing emotional state of a peer. Children of 3 ages--5, 8, and 12 years of age--viewed a picture of a young child actually experiencing happiness, sadness, anger, or in a neutral state. In a portion of instances, information was also provided about the social or nonsocial experience that led to the target child's state. The strategies children nominated fell into a small set of agonistic (nurturant) and antagonistic (aggressive) behaviors intended to change positive or negative states. With increasing age, children tended to nominate a greater proportion of verbal strategies, social strategies (with the exception of strategies nominated to change anger), and strategies that directly addressed the cause of another's emotional state.