A safety assessment of fixed combinations of acetaminophen and acetylsalicylic acid, coformulated with caffeine.

Overuse and abuse of phenacetin-containing mixed analgesics has contributed to end-stage renal disease. Combination analgesics, especially those coformulated with caffeine, have been implicated as imparting a greater risk of analgesic-associated nephropathy (AAN) than single or coformulated analgesics without caffeine. This has led to a recommendation that the sale of "two plus caffeine" analgesic mixtures be reclassified from over-the-counter to prescription only availability. There is a rational basis for coformulating acetylsalicylic acid (ASA) and acetaminophen (paracetamol) as this reduces the dose of each, without altering efficacy. The coformulation of caffeine with these analgesics has a significant adjuvant effect and increases analgesic efficacy 1.4-1.6-fold. Currently available animal and human data do not support the notion that the nephrotoxic risk from coformulated ASA and acetaminophen is higher than the risk from either ASA or acetaminophen alone, in equivalent analgesic doses. There are no epidemiological data that implicate caffeine in AAN, and only limited evidence that links excessive acetaminophen usage to renal disease. There is no evidence that caffeine increases analgesics papillotoxicity directly. The presence of caffeine in mixtures of analgesics are no more addictive than other sources of caffeine. There is no evidence to suggest that adding caffeine to analgesic mixtures enhances the potential for promoting analgesic misuse in the general population. Thus distinct therapeutic benefits of ASA, acetaminophen and caffeine appear to outweigh any known risk. It is doubtful if preventing the availability of these products will significantly affect the role of analgesic abuse/overuse in end-stage renal disease. Better risk management would come from a focused educational program, developed in a close collaboration between industry, healthcare professionals and consumer organizations, such a program must warn against the potential dangers of analgesic and non-steroidal anti-inflammatory drug misuse.

Erythritol: an interpretive summary of biochemical, metabolic, toxicological and clinical data.

A critical and comprehensive review of the safety information on erythritol was undertaken. Numerous toxicity and metabolic studies have been conducted on erythritol in rats, mice and dogs. The toxicity studies consist of long-term feeding studies conducted to determine carcinogenic potential, intravenous and oral teratogenicity studies to determine the potential for effects on the foetus, oral studies in which erythritol was administered over one or two generations to determine the potential for reproductive effects, and studies in bacterial and mammalian systems to determine mutagenic potential. The majority of the safety studies conducted were feeding studies in which erythritol was mixed into the diet at concentrations as high as 20%. The metabolic studies in animals have shown that erythritol is almost completely absorbed, not metabolized systemically and is excreted unchanged in the urine. The safety studies have demonstrated that erythritol is well tolerated and elicits no toxicological effects. The clinical program for erythritol involved a series of single-dose and repeat-dose, short-duration studies which have been used to investigate the human correlates to the physiological responses seen in the preclinical studies. The clinical studies showed erythritol to be well tolerated and not to cause any toxicologically relevant effects, even following high-dose exposure. Erythritol administered orally to humans was rapidly absorbed from the gastrointestinal tract and quantitatively excreted in the urine without undergoing metabolic change. At high oral doses, urinary excretion accounted for approximately 90% of the administered dose with minimal amounts appearing in the faeces. A comparison of the human and animal data indicated a high degree of similarity in the metabolism of erythritol and this finding supports the use of the animal species used to evaluate the safety of erythritol for human consumption. It can be concluded, based on the available studies that erythritol did not produce evidence of toxicity.

The role of transport in chemical nephrotoxicity.

Various physiologic factors play a role in determining the extent of chemical-induced nephrotoxicity. One such factor relates to the transport systems that exist in the kidney. Several examples can be given of organic substances that are nephrotoxic only after being transported into renal tubular cells. Some of the cephalosporin antibiotics have been shown to produce proximal tubular necrosis after transport into those cells. Blockade of transport by competitors eliminates or reduces the nephrotoxic response. Citrinin, a secondary product of fungal metabolism, also produces proximal tubular necrosis, but only after transport into proximal tubular cells. Both the cephalosporins and citrinin utilize the organic anion transporter for entry into the cells, a transporter present in adult animals of all species and probably important physiologically for moving metabolic substrates into cells. Various glutathione conjugates (e.g., S-(1,2-dichlorovinyl) glutathione [DCVG]) also are transported into proximal tubular cells with a resulting nephrotoxicity. DCVG utilizes the sodium-dependent transport process that moves glutathione into proximal tubular cells, a process that is inhibited by probenecid. Finally, certain heavy metals also are transported into renal tubular cells. For example, mercuric ion enters proximal cells both from the luminal and peritubular sides and sulfhydryl compounds modify the transport. Movement of mercury from the peritubular side of the cell may be modified by certain organic anions. The characteristics of these mechanisms are less well understood than the mechanisms for the organic compounds.

Sex differences in monochloroacetate pretreatment effects on chloroform toxicity in rats.

Previous studies have shown that dichloroacetate and trichloroacetate increase the toxicity of CHCl3. The present experiments were designed to determine if monochloroacetate (MCA) similarly affects CHCl3 toxicity. There were occasional differences, but overall kidney function indices (urine volume, osmolality and electrolyte concentration, glucosuria, retention of urea nitrogen in plasma) were not affected differently at either 24 or 48 hr after CHCl3 in saline and MCA pretreated Sprague-Dawley rats of either sex. Males pretreated with MCA had 45-fold greater plasma alanine aminotransferase (ALT) compared to the saline pretreated group similarly dosed with CHCl3. ALT was increased threefold in female rats, a modest change that suggests hepatic damage, and BUN was nonsignificantly increased. Therefore hepatic and renal functions were assessed in females. MCA pretreatment did not alter the effects of CHCl3 on hepatic excretory function or glomerular or tubular function. Bile production and glomerular filtration were both decreased in the MCA group treated with peanut oil, suggesting that MCA impairs both liver and kidney function in female rats. MCA pretreatment increases CHCl3 hepatoxicity markedly in male rats and only slightly in female rats. This difference is likely due to the different effects, in males and females, of MCA on the cytochrome P450 isoforms that activate CHCl3. The effects of MCA on renal function in females would decrease CHCl3 delivery to kidney cells, suggesting that MCA may alter the distribution of CHCl3.

Effects of mercuric chloride on renal plasma membrane function after depletion or elevation of renal glutathione.

The role of renal nonprotein sulfhydryls (NPSH) in mercuric chloride-induced nephrotoxicity has been studied in various laboratories. Similarly, the importance of NPSH for mercuric ion accumulation by renal tissue also has been studied. In this study the potential role of NPSH was examined with respect to mercuric ion effects on membrane transport utilizing isolated membrane vesicles prepared from Sprague-Dawley rat kidneys. Sodium gradient-driven p-aminohippurate (PAH) transport in basolateral vesicles and glucose transport in brush border vesicles were studied. Depletion of NPSH, primarily glutathione (GSH), appeared to alter PAH but not glucose transport. HgCl2 (1 mg/kg) had no effect on either transport system in vesicles isolated from kidneys with normal GSH content, but it markedly disrupted both PAH and glucose transport in vesicles isolated from GSH-depleted rats. The most consistent effects were observed after GSH depletion with diethyl maleate plus buthionine sulfoximine. Elevation of renal GSH by administration of glutathione monoethyl ester blocked the effect of mercuric chloride (4 mg/kg) on glucose transport reported earlier. These data indicate that renal sulfhydryls not only modulate the effects of mercuric chloride, but they also may be important for normal physiological functioning of the PAH transport system.

The effects of potassium chromate and citrinin on rat renal membrane transport.

Both chromate and citrinin have been shown to produce acute renal damage. Although both substrates act on the proximal tubule in the rat, they affect different parts of that nephron segment. As with most nephrotoxicants, the mechanism(s) or subcellular target(s) for citrinin or chromate is unknown. The availability of methodology for isolation of functional membrane vesicles has afforded the opportunity to study the plasma membrane as a target for the effects of citrinin and chromate. Whether studied solely with in vitro conditions or after administration to the rat, chromate exhibited its primary action on the basolateral (BL) membrane vesicles. This was exhibited by a reduction in the p-aminohippurate (PAH) overshoot. At both 3 and 16 hr after treatment (40 mg/kg, sc) there was a significant, but relatively modest, effect on glucose transport by brush border (BB) vesicles. Citrinin, when studied in vitro, inhibited PAH transport (BL vesicles), but had only equivocal effects on BB glucose transport. However, after pretreatment of the rats with citrinin (60 mg/kg, ip), both BL and BB membrane vesicle function was reduced markedly at 3 hr. By 16 hr, an overshoot had returned for both transport substrates, although the glucose overshoot was still significantly below control. These data demonstrate that both citrinin and chromate alter proximal tubular cell membrane function and do so relatively early after administration to the rat. This effect suggests that alteration of membrane function by these nephrotoxicants is an early, if not initiating, event in the production of acute tubular necrosis.

The effects of mercuric chloride on transport by brush border and basolateral membrane vesicles isolated from rat kidney.

Both brush border and basolateral membrane vesicles were prepared from rat kidney by Percoll gradient centrifugation. The addition of mercuric chloride (100 nM) to vesicles prepared from healthy, male, Sprague-Dawley rats reduced p-aminohippurate (PAH) transport by basolateral vesicles. No effect was observed on glucose transport by brush border vesicles even at mercuric chloride concentrations as high as 10 microM. However, when the metal salt was added in the presence of 5% bovine serum albumin, basolateral PAH transport was unaffected. Transport studies also were done with vesicles isolated from rats pretreated with mercuric chloride (4 mg/kg, sc). Transport of PAH was unaffected at all times studied. Glucose transport was unaffected at 1 and 3 hr, but at 16 hr was reduced significantly. By 48 hr, brush border glucose transport had recovered. These data demonstrate that mercuric chloride can alter renal membrane function, and that the effects depend on the membrane vesicle population used. With pretreatment studies, the time after treatment also influences whether or not an effect is seen.

Effect in the rat of the interaction of dichloromaleic acid and carbon tetrachloride on renal and hepatic function.

Water purification generates a variety of chlorinated contaminants, one of which is dichloromaleic acid (DCMA). Exposure to this compound is likely to occur in combination with other drinking water pollutants, some of which are hepatotoxic. This study was designed to examine the interactive effects of carbon tetrachloride (CCl4), a known hepatotoxin, with DCMA on liver and kidney function in the Sprague-Dawley rat. Administration of a single dose of DCMA (200-400 mg/kg, ip) caused modest dose-dependent increases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and plasma urea nitrogen, as well as a marked depletion of nonprotein sulfhydryls (NPSH) in the liver, but not the kidney, by 24 hr. Pretreatment with inducers (phenobarbital or 3-methylcholanthrene) or an inhibitor (SKF 525A) of cytochrome P-450 activity failed to alter the response observed with DCMA alone. Alterations in 24-hr urine volume, osmolality, and water consumption also were observed. DCMA-mediated changes in plasma urea nitrogen and NPSH were reduced in magnitude with coadministration of CCl4 (1 ml/kg, ip), while anticipated CCl4-induced increases in ALT and AST were reduced with coexposure to DCMA. Renal slice experiments indicated that DCMA-treated rats were less able to accumulate the organic anion p-aminohippurate (PAH), whereas DCMA had no effect on accumulation of the organic cation tetraethylammonium (TEA). The combination of CCl4 and DCMA produced only additive effects on organic ion accumulation. These results suggest hepatic interaction possibly related to the metabolism of CCl4 and DCMA, resulting in renal and hepatic toxicity diminished from that observed with exposure to either agent alone.

The effect of combined treatment with potassium dichromate and maleic acid on renal function in the rat.

Subthreshold doses of potassium dichromate (K2Cr2O7) have been shown to enhance or potentiate the nephrotoxic effects of mercuric chloride and citrinin. The mechanism of this effect is unknown. The purpose of this study was to examine the phenomenon further by investigating the interaction of K2Cr2O7 with maleic acid, a nephrotoxicant with an action though to be different from those above. Male rats were housed in stainless steel metabolism cages and received a single intraperitoneal injection of maleic acid (300 mg/kg). K2Cr2O7 (10 mg/kg s.c.) or the combination of maleic acid plus K2Cr2O7. While treatment with either agent alone produced only minimal alterations in renal function, the combination of agents produced marked changes in 24 h urine volume, glucose excretion and osmolality. Water consumption was unaffected by the combined treatment. A marked decline in the capacity of renal slices to accumulate both organic anions (p-aminohippurate) and cations (tetraethylammonium) was observed after treatment with both substances. Modifications in renal and hepatic non-protein sulfhydryl content also were observed. These results suggest that K2Cr2O7, as was observed with both mercury and citrinin, enhanced or potentiated the nephrotoxic effects of maleic acid, probably in the proximal tubule, the principal site of glucose reabsorption and organic ion secretion.

Alterations in the renal function of male and female rats exposed to maleic acid, dichloromaleic acid, and both compounds.

Maleic acid (MA), a known nephrotoxicant in experimental animals, and its chlorinated derivative dichloromaleic acid (DCMA) are present in urban drinking water supplies as by-products of the chlorination process. This study was designed to characterize the effects of simultaneous exposure of subtoxic doses of DCMA and MA on renal function in both sexes of the Sprague-Dawley rat. Urine was collected at 24-h intervals from rats housed individually in stainless steel metabolism cages. Subcutaneous administration of MA at a dose of 150 mg/kg had no effect on several parameters of renal function in either sex at 24 h and only modest effects at 48 h. Renal slice studies showed that treatment of both male and female rats with DCMA (300 mg/kg) reduced p-aminohippurate (PAH) accumulation at 24 h with no effect on the uptake of tetraethylammonium ion (TEA). The combination of MA + DCMA caused a depression of TEA accumulation by slices from the female. Also, changes in urinary glucose excretion and blood urea nitrogen, although additive in the male following coexposure, appeared synergistic or potentiated in the female. These results suggest an enhanced susceptibility of the female rate to the nephrotoxic action of combined exposure to MA and DCMA.

Potential involvement of renal transport mechanisms in nephrotoxicity.

Among the various physiological factors involved in the development of a nephrotoxic insult, certain renal transport systems may be important. The movement of exogenous organic anions and cations from the blood to the tubular fluid is well recognized. The anion transport system, which has been extensively characterized for the transport of p-aminohippuric acid, may have particular relevance. The nephrotoxic effect of citrinin, certain cephalosporin antibiotics and cysteine conjugates in rats and in isolated renal cells can be blocked by probenecid, a drug known to block the organic anion transporter competitively. The reduction in toxic response is correlated with the renal and cellular content of the nephrotoxic chemical.

Unilateral nephrectomy in the rat: effects on mercury handling and renal cortical subcellular distribution.

As unilateral nephrectomy (UNX) is associated with enhanced mercuric chloride nephrotoxicity, studies were undertaken to characterize the effects of UNX on the tissue content, urinary excretion, and renal cortical subcellular distribution of mercury in the rat. Animals were studied immediately, 2 days or 14 days following UNX, during separate phases of compensatory renal hypertrophy. As compared to sham surgery controls, mercury content in renal cortex was higher in all UNX groups at 24 hr following injection and in the immediate and 2-day groups at 1 or 3 hr. However, UNX was not associated with any alteration in mercury content within outer or inner medulla, liver, plasma, or red blood cells. Subcellular distribution studies demonstrated that cytosolic mercury was uniformly elevated in all UNX groups at 1, 3, and 24 hr following injection while mercury bound to "metallothionein-like" proteins or free in the cytosol was increased only at 1 or 3 hr. Nuclear, mitochondrial, or microsomal mercury content was elevated in the animals studied immediately or 14 days after UNX at 3 or 24 hr following injection, while animals studied 2 days after UNX demonstrated a nearly uniform increase at 1, 3, and 24 hr. Single-kidney urinary mercury excretion was elevated in all UNX groups while excretion per gram kidney weight was increased only in the animals studied immediately or 2 days after surgery. These studies suggest that all phases of compensatory renal hypertrophy are associated with an enhanced content of mercury within the cell cytoplasm and in critical cellular organelles, which may explain the enhanced nephrotoxicity seen following UNX.

Epinephrine and norepinephrine enhance p-aminohippurate transport into basolateral membrane vesicles.

The effect of exogenous l-norepinephrine (NE) and l-epinephrine (EP) on transmembrane transport of p-aminohippurate (PAH) was studied in rat proximal tubular basolateral membrane vesicles. A gradient of 50 mM Na+ (out greater than in) and preloading of vesicles with unlabeled PAH were utilized to promote the influx of [3H]PAH into the vesicles. At final concentrations of 1 microM, NE and EP each produced significant elevations in vesicle uptake of [3H]PAH. The enhancement of PAH transport by NE or EP was inhibited by either phentolamine (100 microM) or yohimbine (100 microM). Prazosin (100 microM) or atenolol (100 microM) were unable to inhibit the response to NE. Similarly, prazosin or propranolol (100 microM) were unable to inhibit the response to EP. Clonidine (1 microM) also produced a significant elevation of PAH uptake, an effect inhibited by both phentolamine and yohimbine. Basolateral Na+-K+-adenosine triphosphatase activity also was increased significantly by either NE or EP (1 microM). Both agonists produced significant elevations of PAH uptake into vesicles preloaded with ATP. However, in the absence of NE or EP, PAH uptake into ATP-loaded vesicles was not significantly greater than into control vesicles. It was concluded that NE and EP enhance Na+-coupled PAH transport and that this effect may be mediated by alpha-2 adrenergic receptors. Activation of Na+-K+-adenosine triphosphatase is a possible mechanism whereby adrenergic agonists may exert effects on Na+-coupled transport across the basolateral membrane.

Characterization of adrenergic receptors on proximal tubular basolateral membranes.

Alpha adrenergic receptors are identified on basolateral plasma membranes derived from proximal tubular epithelial cells. The density of alpha 2 receptors was over two-fold greater than alpha 1 receptors. The basolateral membranes were devoid of beta receptors. These results support previous demonstrations of alpha adrenergic receptors in rat renal cortex and concur with studies which suggest a limited presence of beta receptors on rat proximal tubules.

The effect of chromate on citrinin-induced renal dysfunction in the rat.

Previous studies in this laboratory revealed an effect of chromate to potentiate the nephrotoxic effects of mercuric ion. Citrinin, an organic anion, is a known nephrotoxin. The present study was undertaken to assess the possible interaction of chromate and citrinin on renal function. Male Sprague-Dawley rats were housed in metabolism cages and injected with citrinin (35 mg/kg), chromate (10 mg/kg) or the combination. The combination of nephrotoxicants caused an increased excretion of urine greater than the sum of the individual responses. A similar response was observed with urinary glucose concentrations and glucose excretion without changes in blood glucose levels. These data indicate that chromate can potentiate the nephrotoxic action of citrinin in the rat.

The effect of unilateral nephrectomy on the nephrotoxicity of mercuric chloride in the rat.

Unilateral nephrectomy (UNX) induces a dramatic change in single-kidney structure and function. Therefore, the effects of nephrotoxins may be altered. To evaluate this possibility, mercuric chloride (2 mg/kg, sc) was given to male, Sprague-Dawley rats 2 days following either UNX or sham surgery. Nonoliguric acute renal failure developed and was qualitatively similar in both groups. Glomerular filtration rate (GFR) reached a nadir on Day 2 and was reduced to a greater extent in the UNX group. Furthermore, recovery of GFR was slower and occurred to a lesser extent by Day 10 in the animals subjected to UNX. Evidence of significant tubular dysfunction was present during the acute phase in both groups, as reflected by changes in the fractional excretion of sodium or lysozyme. Persistent tubular dysfunction was noted on Day 10 in both the sham and UNX groups, but the degree of dysfunction was greater in the UNX animals. The in vitro uptake of organic ions by renal cortical slices was reduced 24 hr following the injection of mercuric chloride although no difference was seen between the experimental groups. Mercury content within renal cortex was not increased in the UNX group at 1 or 3 hr but was higher 24 hr postinjection. Total urinary mercury excretion during the first day was not altered by UNX although single-kidney excretion was increased dramatically. These studies suggest that rats are more susceptible to mercuric chloride-induced nephrotoxicity 2 days following UNX. Although the mechanism(s) of this enhanced injury remains unclear, it does not appear to be completely related to an increase in renal cortical mercury content.

The effect of depletion of nonprotein sulfhydryls by diethyl maleate plus buthionine sulfoximine on renal uptake of mercury in the rat.

Rats pretreated with diethyl maleate (DEM, 3.37 mmol/kg, ip) and buthionine sulfoximine (BSO, 0.45 mmol/kg, ip) and subsequently given mercuric chloride (HgCl2, 0.014 mmol/kg, sc) had a significantly greater mortality rate over the 24 hr after injection than rats given only HgCl2 or HgCl2 following either DEM or BSO alone. Depletion of nonprotein sulfhydryls (NPSH) in the kidney significantly decreased mercury uptake in that organ. A similar effect was not seen in the liver despite marked depletion of NPSH. Similarly, there was a tendency for less in vitro mercury accumulation in renal cortical slices from rats made glutathione deficient by DEM + BSO compared to control, or rats made glutathione deficient by DEM or BSO alone. Depletion of nonprotein sulfhydryls by the combination of the depleting agents diethyl maleate plus buthionine sulfoximine (DEM + BSO) had a greater effect to alter organic ion accumulation in renal cortical slices than the agents alone. The higher mortality produced by mercuric chloride after DEM + BSO pretreatment may have been due to an increased availability of mercury in lethal concentrations at other organ sites. These data suggest the possible importance of NPSH in renal mercuric ion accumulation, but not in the liver.