The renal mitochondrial toxicity of beta-lactam antibiotics: in vitro effects of cephaloglycin and imipenem

The nephrotoxic beta-lactam antibiotics cephaloridine, cephaloglycin, and imipenem produce irreversible injury to renal mitochondrial anionic substrate uptake and respiration after 1 to 2 h of in vivo exposure. Toxicity during in vitro exposure is nearly identical but is immediate in onset and is reversed by the mitochondria being washed or the substrate concentrations being increased. A model of injury that accounts for these findings proposes that the beta-lactams fit carriers for mitochondrial substrate uptake, causing inhibition that is initially reversible and becomes irreversible as the antibiotics acylate the transporters. These studies were designed to create an environment of prolonged in vitro exposure, first, to determine whether toxicity becomes irreversible with time and, second, to study the molecular properties of toxicity. Respiration with and the uptake of succinate and ADP were measured in rabbit renal cortical mitochondria exposed for 2 to 6 h to 300 to 3,000 micrograms of cephalexin (nontoxic) or cephaloglycin or imipenem (nephrotoxic) per mL and then washed to remove the antibiotic. In vitro cephalexin reduced respiration only slightly and was therefore not studied further. Cephaloglycin and imipenem irreversibly reduced both respiration and succinate uptake. ADP uptake was unaffected by cephaloglycin and was slightly reduced by imipenem. Finally, cilastatin, which prevents the tubular necrosis produced by imipenem in vivo, reduced its mitochondrial toxicity in vitro. It is concluded that the pattern of in vitro injury of the nephrotoxic beta-lactams to mitochondrial substrate uptake and respiration evolves in a time-dependent and concentration-dependent manner, consistent with the proposed model of acylation and inactivation of substrate transporters, and that the protective action of cilastatin against imipenem occurs at least partly at a subcellular level.     

Effects of anti-lipid A human monoclonal antibody on lipopolysaccharide-induced toxicity to the kidney

Studies were done to evaluate the effects of the human monoclonal anti-lipid A IgM antibody A6(H4C5) on several components of the hemodynamic and renal toxicity of the cell wall lipopolysaccharide of E. coli 0111:B4. Antibody (0.25 to four mg./kg. BW) was administered 0.5 hour before, or premixed for one hour with, lipopolysaccharide (0.05 mg./kg., a 14 to 18% lethal dose), and the following measurements made over 0.5 to 3.5 hours of study: systemic arterial blood pressure, renal plasma flow, and glomerular filtration. The proximal tubular cell cytotoxicity of 90 mg./kg. of the cephalosporin cephaloridine was also quantified in similarly treated animals sacrificed 48 hours later. While one mg./kg. of antibody prevented the reduction by the lipopolysaccharide of renal plasma flow, it did not prevent the nephrotoxic synergy with cephaloridine, and four times the antibody dose did not prevent lipopolysaccharide-induced hypotension or reduced glomerular filtration. These amounts of this antibody protect leukopenic rabbits against the lethality of the slow onset bacteremic model of Pseudomonas conjunctivitis. It is suggested that the incompleteness of protection in this study may be the result of the sensitivity of the assay methods used and/or the acute endotoxemia produced in these animals.     

Nephrotoxic drugs

The nephrotoxic effects of cyclosporine, aminoglycoside antibiotics, cisplatin, amphotericin B, beta-lactam antibiotics and indomethacin are reviewed. These drugs were chosen because they are among the most frequent causes of renal injury in children. In addition, their nephrotoxicity is caused by different mechanisms. Several generalizations can be made, however. First, agents which cause tubular damage tend to be synergistic in their toxic effects. This synergism is seen when several nephrotoxic drugs are given simultaneously. In addition, the use of a nephrotoxic agent in a patient with pre-existing renal disease can result in severe tubular injury. Second, serum levels of the drug frequently fail to correlate with the degree of nephrotoxicity in individual patients. Third, early signs of renal injury can be subtle (e.g., minor changes in electrolyte excretion) or dramatic (e.g., acute renal failure). The sublte changes are particularly important, since they can be useful predictors of serious nephrotoxicity.     

Renal tubular necrosis following cephalothin.

Renal tubular necrosis was observed following intramuscular injections of cephalothin into rats. Lesions were consistently produced with 5.0g/kg and were maximal in severity at the 2nd and 3rd days following injection. Renal tubular necrosis following cephalothin was similar in morphology to that produced by nephrotoxic doses (2.0 g/kg) of cephaloridine. The nephrotoxic potential of cephalothin has been demonstrated in the rat model and caution is urged in using large doses of cephalothin.     

CEPHALORIDINE PROPHYLAXIS IN RESECTION OF THE LARGE INTESTINE

A controlled prospective clinical trial of cephaloridine chemoprophylaxis in resection of the large intestine was undertaken between 1974 and 1978. Data were available on 159 of 177 unselected patients. All were operated on by one surgeon. Three groups were studied: intraabdominal resection and anastomosis (102 patients); pullthrough resection and anastomosis (30 patients): and resection, with colostomy or ileostomy, without anastomosis (27 patients). In the total patient series cephaloridine reduced wound infection from 38.3% to 15.4% (P< 0.003). There was no significant decrease in intraabdominal infection. In the group of patients undergoing intraabdominal resection and anastomosis the wound infection rate was reduced from 40.0% to 14.9% (P< 0.01). Cephaloridine reduced wound infection from 50.0% to 21.4% (P = 0.05) in those patients in whom drainage tubes were inserted. A decrease in the incidence of faecal fistula from 10.9% to 4.3% was not significant. Wound infections were not reduced significantly after pullthrough excisions or resections without anastomosis. The results support the routine prophylactic use of cephalosporins in patients undergoing intraabdominal resection of the large intestine with anastomosis.     

Cephaloridine prophylaxis in resection of the large intestine.

A controlled prospective clinical trial of cephaloridine chemoprophylaxis in resection of the large intestine was undertaken between 1974 and 1978. Data were available on 159 of 177 unselected patients. All were operated on by one surgeon. Three groups were studied: intraabdominal resection and anastomosis (102 patients); pullthrough resection and anastomosis (30 patients): and resection, with colostomy or ileostomy, without anastomosis (27 patients). In the total patient series cephaloridine reduced wound infection from 38.3% to 15.4% (P less than 0.003). There was no significant decrease in intraabdominal infection. In the group of patients undergoing intraabdominal resection and anastomosis the would infection rate was reduced from 40.0% to 14.9% (P less than 0.01). Cephaloridine reduced wound infection from 50.0% to 21.4% (P = 0.05) in those patients in whom drainage tubes were inserted. A decrease in the incidence of faecal fistula from 10.9% to 4.3% was not significant. Wound infections were not reduced significantly after pullthrough excisions or resections without anastomosis. The results support the routine prophylactic use of cephalosporins in patients undergoing intraabdominal resection of the large intestine with anastomosis.     

Role of the Renal Cysteine Conjugate [Greek small letter beta]-Lyase Pathway in Inhaled Compound A Nephrotoxicity in Rats

Background: The sevoflurane degradation product compound A is nephrotoxic in rats and undergoes metabolism to glutathione and cysteine S-conjugates, with further metabolism by renal cysteine conjugate [Greek small letter beta]-lyase to reactive intermediates. Evidence suggests that toxicity is mediated by renal up-take of compound A S-conjugates and metabolism by [Greek small letter beta]-lyase. Previously, inhibitors of the [Greek small letter beta]-lyase pathway (aminooxyacetic acid and probenecid) diminished the nephrotoxicity of intraperitoneal compound A. This investigation determined inhibitor effects on the toxicity of inhaled compound A.

Methods: Fischer 344 rats underwent 3 h of nose-only exposure to compound A (0 - 220 ppm in initial dose - response experiments and 100 - 109 ppm in subsequent inhibitor experiments). The inhibitors (and targets) were probenecid (renal organic anion transport mediating S-conjugate uptake), acivicin ([Greek small letter gamma]-glutamyl transferase), aminooxyacetic acid (renal [Greek small letter beta]-lyase), and aminobenzotriazole (cytochrome P450). Urine was collected for 24 h, and the animals were killed. Nephrotoxicity was assessed by histology and biochemical markers (serum BUN and creatinine; urine volume; and excretion of protein, glucose, and [Greek small letter alpha]-glutathione-S-transferase, a predominantly proximal tubular cell protein).

Results: Compound A caused dose-related proximal tubular cell necrosis, diuresis, proteinuria, glucosuria, and increased [Greek small letter alpha]-glutathione-S-transferase excretion. The threshold for toxicity was 98 - 109 ppm (294 - 327 ppm-h). Probenecid diminished (P < 0.05) compound A-induced glucosuria and excretion of [Greek small letter alpha]-glutathione-S-transferase and completely prevented necrosis. Aminooxyacetic acid diminished compound A - dependent proteinuria and glucosuria but did not decrease necrosis. Acivicin increased nephrotoxicity of compound A, and aminobenzotriazole had no consistent effect on nephrotoxicity of compound A.     

Cellular and molecular aspects of drug transport in the kidney

The kidney plays an important role in the elimination of numerous hydrophilic xenobiotics, including drugs, toxins, and endogenous compounds. It has developed high-capacity transport systems to prevent urinary loss of filtered nutrients, as well as electrolytes, and simultaneously to facilitate tubular secretion of a wide range of organic ions. Transport systems for organic anions and cations are primarily involved in the secretion of drugs in renal tubules. The identification and characterization of organic anion and cation transporters have been progressing at the molecular level. To date, many members of the organic anion transporter (OAT), organic cation transporter (OCT), and organic anion-transporting polypeptide (oatp) gene families have been found to mediate the transport of diverse organic anions and cations. It has also been suggested that ATP-dependent primary active transporters such as MDR1/P-glycoprotein and the multidrug resistance-associated protein (MRP) gene family function as efflux pumps of renal tubular cells for more hydrophobic molecules and anionic conjugates. Tubular reabsorption of peptide-like drugs such as beta-lactam antibiotics across the brush-border membranes appears to be mediated by two distinct H+/peptide cotransporters: PEPT1 and PEPT2. Renal disposition of drugs is the consequence of interaction and/or transport via these diverse secretory and absorptive transporters in renal tubules. Studies of the functional characteristics, such as substrate specificity and transport mechanisms, and of the localization of cloned drug transporters could provide information regarding the cellular network involved in renal handling of drugs. Detailed information concerning molecular and cellular aspects of drug transporters expressed in the kidney has facilitated studies of the mechanisms underlying renal disposition as well as transporter-mediated drug interactions.     

Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation

Aristolochic acid contamination in herbal remedies leads to interstitial fibrosis, tubular atrophy, and renal failure in humans. To study the cellular mechanisms contributing to the pathophysiology of this renal disease, we studied Wistar rats treated with aristolochic acid and measured tubular and interstitial cell proliferation, epithelial/mesenchymal cell marker expression, tubular membrane integrity, myofibroblast accumulation, oxidative stress, mitochondrial damage, tubular apoptosis, and fibrosis. Oxidative stress, a loss of cadherin concomitant with vimentin expression, basement membrane denudation with active caspase-3 expression, and mitochondrial injury within tubular cells were evident within 5 days of administration of the toxin. During the chronic phase, interstitial mesenchymal cells accumulated in areas of collagen deposits. Impaired regeneration and apoptosis of proximal tubular cells resulted in tubule atrophy with a near absence of dedifferentiated cell transmembrane migration. We suggest that resident fibroblast activation plays a critical role in the process of renal fibrosis during aristolochic acid toxicity.     

Effect of chronic cisplatin administration on phosphate and glucose transport by the renal brush border membrane

Cisplatin (CIS-diamine dichloroplatinum) is a highly nephrotoxic antineoplastic agent which may cause acute renal failure and renal tubular dysfunction. In the present study we have examined the effect of chronic cisplatin administration on sodium-dependent 32P-phosphate and 3H glucose transport by the renal brush border membrane vesicles (BBMV). Our results indicate that both transport mechanisms were significantly reduced at the BBMV following cisplatin therapy due to an increased Km (0.13 +/- 0.09 vs. 0.34 +/- 0.09 mM; p = less than 0.01) without significant change in Vmax (56 +/- 18 vs. 44 +/- 17 pM/mg/s). The results of these studies indicate that cisplatin causes a diffuse renal injury in the proximal segment of the nephron altering both transport mechanisms. Possible mechanisms of cisplatin nephrotoxicity are discussed.     

Nephrotoxicity induced by cancer chemotherapy with special emphasis on cisplatin toxicity

Renal failure in cancer patients is a common problem in oncology; this complication is frequently multifactorial in origin. Several antineoplastic agents are potentially nephrotoxic; previous renal impairment as well as combinations with other nephrotoxic drugs may increase the risk of nephrotoxicity during administration of chemotherapy. Methotrexate-related renal damage most frequently occurs with high-dose therapy and can be avoided by forced alkaline diuresis and administration of folinic acid. Renal dysfunction secondary to semustine (CH3-CCNU) is clearly related to cumulative doses in excess to 1,200 mg/m2; the onset may be delayed and renal failure progress despite drug discontinuation. Streptozotocin is also nephrotoxic and may cause proteinuria and renal tubular acidosis; progressive renal failure can be predicted by a close monitoring of proteinuria and prevented by drug discontinuance. Mitomycin-associated renal failure frequently presents with signs of microangiopathic hemolytic anemia; renal failure is usually delayed but occasionally, it may be rapidly progressive despite drug discontinuance. Cisplatin nephrotoxicity is clearly dose-related and used to be considered dose limiting. Renal insufficiency can be prevented by hydration and forced diuresis; in addition, hyperhydration with mannitol-induced saline diuresis may allow administration of high doses and thus circumvent the dose-limiting effect of cisplatin-induced renal toxicity. Cisplatin-induced renal magnesium wasting occurs frequently and should be supplemented. Other approaches to reduce cisplatin nephrotoxicity are currently under investigation and are discussed.     

Hemorrhagic risk factors during beta-lactam antibiotics therapy

Any type hemorrhagic manifestation may occur 12 hours to 5 weeks after the administration of beta-lactam antibiotics. The mechanisms of blood losses proved to be by: 1) immunologic thrombocytopenia (penicillins); 2) alteration of platelet functions (semisynthetic penicillins); 3) hypoprothrombinemia (cephalosporins). The risk factors for the occurrence of hemorrhage under beta-lactam antibiotics therapy are: concomitant administration of cytostatics for a neoplastic malignancy; b) acute or chronic renal failure; c) concomitant treatment for duodenal and gastric ulcer; d) malnutrition; e) dosage and duration of antibiotic administration. The frequency of bleeding under beta-lactams is not determined as yet. A severe case diagnosed at the IIIrd Medical Clinic of Iasi presenting spontaneous daily nasal bleedings that occurred 24 hours after the initiation of the treatment with cephalosporins (Kefurox) is presented. In this patient the risk factor was chronic renal failure.     

Interstitial nephritis and nephrogenic diabetes insipidus in a patient treated with pemetrexed

We present a case of interstitial nephritis and nephrogenic diabetes insipidus (NDI) in a patient treated with pemetrexed (500 mg/m²) for non-small cell lung cancer. Renal impairment and diabetes insipidus appeared after the first treatment cycle while he totally received four cycles of chemotherapy. There was not any significant myelosuppression and the patient was on regular supplementation with folic acid and vitamin B₁₂. He was not on any other medications and he did not receive any nephrotoxic agents. Kidney biopsy showed acute tubular necrosis together with interstitial inflammatory infiltrate of mononuclear cells and interstitial fibrosis occupying 25% of the cortex. There was not any improvement of renal function after a 2-week trial of oral prednisone. In the present case report, we review the literature for pemetrexed-induced renal toxicity and the possible mechanisms involved.     

Paracetamol-induced renal tubular injury: a role for ER stress

Paracetamol (also known as acetaminophen) causes acute and chronic renal failure. While the mechanisms leading to hepatic injury have been extensively studied, the molecular mechanisms of paracetamol-induced nephrotoxicity are poorly defined. Paracetamol induced cell death with features of apoptosis in murine proximal tubular epithelial cells. While paracetamol increased the expression of the death receptor Fas on the cell surface, the Fas pathway was not involved in the paracetamol-induced apoptosis of tubular cells. The mitochondrial pathway was not activated during paracetamol-induced apoptosis; there was no dissipation of mitochondrial potential or release of apoptogenic factors such as cytochrome c or Smac/DIABLO. However, paracetamol-induced apoptosis is a caspase-dependent process that involves activation of caspase-9 and caspase-3 in the absence of cytosolic cytochrome c or Smac/DIABLO. The authors also detected induction of endoplasmic reticulum (ER) stress, characterized by GADD153 upregulation and translocation to the nucleus, as well as caspase-12 cleavage. Interestingly, after treatment of murine tubular cells with paracetamol and calpain inhibitors, the caspase-12 cleavage product was still detectable, and calpain inhibitors were unable to protect tubular cells from paracetamol-induced apoptosis. The results suggest that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and identify ER stress as a therapeutic target in nephrotoxicity.     

Gentamicin-induced Bartter-like syndrome

Gentamicin is well known to be associated with nephrotoxicity, including acute renal failure and renal tubular dysfunction. A Bartter-like syndrome has also been described as a toxic manifestation of gentamicin therapy in adults, but this nephrotoxic syndrome has not been well characterized in children. In this report we describe the clinical course of four patients with gentamicin-associated Bartter-like syndrome. These patients ranged in age from 4 months to 17 years; they all demonstrated evidence of renal tubulopathy, primarily affecting the distal nephron. Hypocalcemia, hypomagnesemia, alkalosis, and hypokalemia were the main manifestations in these patients. After discontinuation of gentamicin, recovery of the renal tubular functions and resolution of the electrolyte abnormalities were complete in all patients. Received December 10, 1996; received in revised form May 5, 1997; accepted May 14, 1997     

Hypertrophy of renal mitochondria

Compensatory renal hypertrophy leads to an increase in the size and metabolic capacity of renal tubular cells. Increased transport and metabolic activities must be sustained by an augmented rate of energy production, which is largely dependent on mitochondrial processes. Although previous studies have suggested that mitochondria proliferate in the hypertrophying cell, the data to support this have not been convincing. This study was designed to determine whether the mitochondria of the hypertrophied renal proximal tubular cell undergo hypertrophy or proliferation. Flow cytometric analysis of proximal tubular cells obtained from the kidneys of uninephrectomized rabbits revealed an increase in cell size and RNA content compared with control cells but showed no change in DNA content and nuclear size and no evidence of entry into the S/G2/M phases of the cell cycle. Histomorphometric analysis of cortical proximal tubules revealed that although cytoplasmic volume increased, mitochondrial density remained constant, indicating that mitochondrial volume increases in proportion to the increase in cell volume. By day 14, mitochondrial volume had increased 66% above control values. Electron microscopic examination of isolated S2 proximal tubules from 5/6 nephrectomized rabbits with maximal hypertrophy revealed mitochondrial cristae which appeared to be more densely packed than that in normal cells. The size of the functional mitochondrial pool per cell was determined by rhodamine-123 fluorescence. This increased within 24 h of uninephrectomy, peaked at approximately 80% above control levels at 5 days, and remained elevated throughout the 16 days of observation. The initial increase (days 1 and 2) occurred before a measurable increase in mitochondrial volume occurred and presumably reflects an increase in mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tenofovir-induced kidney disease: an acquired renal tubular mitochondriopathy

Tenofovir, used in combination with other antiretroviral agents, is an effective therapy for HIV infection. Although large clinical studies and post-marketing data support a benign renal profile for tenofovir, numerous cases of kidney injury raise concern for nephrotoxic potential. Early human studies and experimental evidence suggested that tenofovir itself was not associated with mitochondrial toxicity within the kidney. However, recent animal data demonstrate that tenofovir causes mitochondrial DNA depletion and mitochondrial toxicity. Herlitz et al. confirm the nephrotoxicity of tenofovir in humans. They describe its clinical consequences, histopathologic findings, and its mitochondrial toxicity in HIV+ patients.     

Quantitating the severity of proximal tubular brush border injury by a simple direct binding radioimmunoassay

The purpose of this investigation was: (1) to establish a simplified radioimmunoassay (RIA) for quantitating renal tubular epithelial antigens (RTE) in urine; (2) to ascertain whether urine RTE concentrations as measured by this technique correlate with the severity of acute nephrotoxic and ischemic injury; and (3) to ascertain whether increased urinary RTE is a specific marker of renal tubular injury. A direct binding RTE RIA was established using 125I labelled anti-RTE antibody and 10% polyethylene glycol to separate bound from unbound anti-RTE 125I. This RIA is simpler than previously described RTE assay methods since: (1) double antibody separation techniques are eliminated; (2) RTE antigen purification from crude proximal tubular fragments is no longer necessary; and (3) immunoglobulin G(IgG) rather than more radiosensitive RTE is used as the radioligand. To test the utility of this assay as a marker of acute tubular injury anesthetized rats were subjected to graded nephrotoxic (HgCl2: 0--20 mg/kg) or bilateral renal ischemic (0--32 min) insults. Glomerular filtration rates (GFR) (clearance iothalamate 125I) and RTE concentrations were measured sequentially. Post-renal injury, RTE concentrations rose above control values and the degree of elevation strongly correlated with the severity of GFR depression (r = 0.72--0.81; p less than 0.02--0.05). The source of this increased urinary RTE was the proximal tubule since brush border loss was demonstrated histologically and because no RTE could be detected in serum. Rats whose GFRs were acutely depressed by inducing either volume depletion or acute experimental glomerulonephritis (nephrotoxic serum nephritis) all had normal urine RTE concentrations. These results suggest that RTE quantitation by this technique may provide a specific and early quantitative index of the severity of acute nephrotoxic and ischemic renal injury.     

A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro

Nephrotoxicity due to drugs and environmental chemicals accounts for significant patient mortality and morbidity, but there is no high throughput in vitro method for predictive nephrotoxicity assessment. We show that primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells rendering them desirable to use in such in vitro systems. To identify a reliable biomarker of nephrotoxicity, we conducted multiplexed gene expression profiling of HPTECs after exposure to six different concentrations of nine human nephrotoxicants. Only overexpression of the gene encoding heme oxygenase-1 (HO-1) significantly correlated with increasing dose for six of the compounds, and significant HO-1 protein deregulation was confirmed with each of the nine nephrotoxicants. Translatability of HO-1 increase across species and platforms was demonstrated by computationally mining two large rat toxicogenomic databases for kidney tubular toxicity and by observing a significant increase in HO-1 after toxicity using an ex vivo three-dimensional microphysiologic system (kidney-on-a-chip). The predictive potential of HO-1 was tested using an additional panel of 39 mechanistically distinct nephrotoxic compounds. Although HO-1 performed better (area under the curve receiver-operator characteristic curve [AUC-ROC]=0.89) than traditional endpoints of cell viability (AUC-ROC for ATP=0.78; AUC-ROC for cell count=0.88), the combination of HO-1 and cell count further improved the predictive ability (AUC-ROC=0.92). We also developed and optimized a homogenous time-resolved fluorescence assay to allow high throughput quantitative screening of nephrotoxic compounds using HO-1 as a sensitive biomarker. This cell-based approach may facilitate rapid assessment of potential nephrotoxic therapeutics and environmental chemicals.     

Amikacin-induced nephropathy: is there any protective way?

Amikacin is a commonly used antibacterial drug that can cause significant nephrotoxic effects in both humans and experimental animals. It has been reported that one mechanism of the toxic effects of aminoglycoside antibiotics are the result of oxidative reactions. The aim of this study is to examine the effects of N-acetylcysteine, a thiol-containing antioxidant, on renal function (serum creatinine) and morphology (renal tubular damage) in mice subjected to amikacin-induced nephrotoxicity. A total of 32 mice were equally divided into four groups that were injected with either saline, amikacin (1.2 g/kg intraperitoneally), N-acetylcysteine (150 mg/kg intraperitoneally for three days) plus amikacin (1.2 g/kg intraperitoneally on the third day as a single dose), or N-acetylcysteine (150 mg/kg intraperitoneally). Amikacin administration led to granulovacuolar tubular degeneration in light microscopic examination and myeloid bodies, mitochondrial electron-dense material deposition, and mitochondrial swelling in the proximal tubule epithelium in the electron microscopic evaluation. N-acetylcysteine administration before amikacin injection caused significant decreases in myeloid body and mitochondrial swelling and granulovacuolar tubular degeneration formation. Serum creatinine levels did not change as a result of any treatment. The results show that N-acetylcysteine has a protective effect on nephrotoxicity induced by amikacin. Higher doses of amikacin should be tried to observe biochemical effects.