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Articles of general interest
467
damage DNA has been exploited to provide a rapid plate test for mutagens, which involves measuring the inhibition of growth of parent and mutant strains (Kada et al. ibid 1974,26, 243; Slater et al. Cancer Res. 1971,31, 970).Longneckeret al. (ibid 1974,34, 1658) evaluated the test with a wide variety ,of mutagens,however, and concluded that it was not as sensitive as was first thought, especiallywith mutagensrequiring metabolic activation. Moreover, the observationthat for somerepair-deficientstrains the extent of killing by mutagensis unrelated to the number of induced mutations detracts somewhat from the value of the method (Hince & Neale,
luated most carefully in the light of the recognized limitations, but such tests, in combination with other short-term tests for mutagenicity including those involving mammaliancells in culture, would provide a most effective primary screen for detecting mutagens.This approach has been suggestedby Bridges(ibid 1974,26, 335) who hasoutlined a threetier systemof mutagenicitytesting,in which this primary screeningmay be followedby in viva testsusing whole mammalsand finally, in the light of the earlier resultsand potential levels of exposure,by experimentsdesignedto give somequantitative indication of the potential risk to man. Obviously, for a full Mutation Res. 1974, 22. 235). definition of the mutagenicpotential of a chemical In spite of the limitations discussedin this article, it is necessaryto determinemany more facts, such microbial mutagenicity testsare obviously the only as the mutagenconcentration at the site of action, meansavailablefor screeninglargenumbersof chemi- the active metabolic products and their rate of calsand drugs for mutagenicactivity, sincein terms absorption,metabolismand elimination. of speedand sensitivity they are vastly superior to in vivo testsin mammals.Clearly, both positive and [I. R. Rowland-BIBRA] negative results from microbial tests must be eva-
RENAL RESPONSE TO LEAD.. . . .
.
The three heavy metals,lead (Pb), mercury and readily than from the lysosomes,since the concadmium,are ingestedin smallamountswith many centration in both organelleswas virtually the same types of food and are currently the object of some 7 and 14daysafter treatment.Furthermore,previous concernto health authoritiesall over the world. Des- oral dosingw.ith stable Pb acetate(300 mg/kg/day) pite wide differencesin their absorptionand excretion for 7 consecutivedays had little effect on the lysosorates and in the pathologicalchangesthey may in- ma1 uptake of ‘03Pb, whereasthe mitochondrial duce, thesemetalspossess at least one common fea- uptake of the isotopewasreducedby 3040%. To someextent, thesebiochemicaldata have been ture, namelytheir ability to accumulatein the kidney and producerenal damage.The type of damagepro- reflected in the findings of an ultrastructural study duced by each element has distinctive features, by Murakami & Hirosawa(Nature, Lmd. 1973,245, although someeffectsare common to all three. This 153),who usedelectron-microscopic autoradiography article is the first of a short serieson thesemetals to demonstratethe presenceof “‘Pb within the mitoand is concernedspecificallywith the renal changes chondria of the tubular epithelial cellsof the kidney associatedwith Pb intoxication. 30 minutesafter administrationof an ip injectionconPb penetratesrapidly into the renal cells.Within taining this radioactive isotope. The mitochondriafrom Pb-treatedanimalsdisplay 1 hour of iv injection of .I00 g *l’Pb as Pb acetate into rats, the Pb was identified in all the fractions other typesof abnormality apart from thesePb incluof a renal homogenate(Castellino& Aloj, Br. J. ind. sions.Usually elongatedand rod-shaped,the mitoMed. 1969,26, 139).During the first 24 hours after chondria of the proximal renal tubulesbecomeoval a singleinjection, the amount of Pb in the nuclear or roundedand increasein volume (Goyer, Lab. Inand mitochondrial fractions increasedat the expense vest. 1968,19, 71).Cristaemay be vesicularbut more of that in the microsomalfraction, and throughout often they are shortenedand marginal,while matrical the subsequent 8 daysof the study the distribution re- granules become less homogeneousand tend to mainedrelatively stable,with considerablyhigherlevels clump. in the two former fractions than in the microMore significant,perhaps,is the informationon the somaland solublefractions.Washingwith 0.25M-SUCfunctional state of the mitochondria obtained by rose,aloneor containing0001M-ethylenediaminetetra- Goyer et al. (ibid 1968,19, 78) in a study designed acetic acid (EDTA), removed Pb lessreadily from to elucidatethe meaningof the observedmorphologithe mitochondrialthan from other fractions, indicat- cal changes.Mitochondria isolatedfrom the proximal ing somedegreeof stable binding. Further insight tubular cellsof rats fed a diet containing 1%Pb acerate into the distribution of Pb within the kidney cells tate for 6 weekswere found to have a decreased has been provided by Barltrop et al. (J. Lab. clin. of respirationand partially uncoupledoxidative phosMed. 1971,77, 705).After injectingradioactive ‘03Pb, phorylation. This functional defect was accentuated theseauthors subdividedthe original mitochondrial by in vitro ageingof the mitochondria at 0°C and fraction of the kidney homogenateinto mitochondrial was partially reversedby addition of EDTA. Moreand lysosomalfractionsand found that 2 hoursafter over, the mitochondrial outer membranes,where treatment the mitochondriacontained four timesas someof the respiratory enzymesare located, were much “‘Pb as the lysosomalfraction. It appears, ruptured. Further evidenceof an impairmentin the however,that Pb is lost from the mitochondriamore respiratory-enzyme‘activity of mitochondria from
468
Articles of general interest
thesePb-intoxicatedrats wasobtainedat a later stage of this work, when a significant reduction in cytochromesuag and h was demonstrated(Rhyne & Goyer, E.uplnzol.Path. 1971,14, 386). This mitochondrial defect may provide some explanation for the amino-aciduriaseenas a result of Pb poisoning,sincethe transport of amino acids in the proximal renal tubule is an active, energyrequiring process(Goyer et al. lot. cit.). The aminoaciduria occurring in Pb poisoninghasbeencharacterized as “generalized”and is usually accompanied by glycosuria and hyperphosphaturia(Chisolm, J. Pediat. 1962,60. 1). It was investigatedfurther by Goyer et al. (Proc. Sot. exp. Biol. Med. 1970,135, 767) who confirmed that in Pb-poisonedrats it is largely secondaryto impaired function of the renal tubules.They demonstrateda one- to threefoldelevation in the renal clearancesof mostamino acids; the increasein histidineclearancewasexceptionallylarge, but changesin glycine and valine clearanceswere slightand tyrosine clearanceactually decreased. This lossof amino acids is unlikely to be due entirely to the impairmentof tubular reabsorption,however,and the formation of metal-amino acid complexesmay be a contributory factor. This excessiveamino-aciduriadevelopswithin 3 weeksin rats fed 1% Pb acetate,and administration of Pb for longerperiodstendsto accentuatethe renal tubular damage.Feedingof 1 or 2% Pb acetate for 10-40weeksresultedin an increasein kidney weight and a decrease in the capacity of the kidney to synthesizeglucoseand metabolizepyruvate (Hirsch, Toxic. appl. Pharmac. 1973,25,84).Excretion of p-aminohippurate during the first 2 hours after administration was higher than that in controls. Feedingof a diet containing 200 ppm Pb or lessdid not affect these parameters.Histologicalexaminationrevealedvacuolar degenerationin the distal segmentof the proximal renal tubulesof rats subjected(either directly or, initially. via the milk of treateddams)to a diet contain-
ing 2% Pb acetate for the first 10 weeksof life. A 4% dietary level of Pb acetate caused,in addition, somecellular necrosis. The renal responseto Pb intoxication has been studiedlessintensively in other speciesthan in the rat, but the data available indicate that broadly speakingsimilar changesoccur. Levels of activity of lactate dehydrogenase and glucose-6-phosphate dehydrogenase were up to twice as high as normal in kidney homogenatesof guinea-pigsgiven Pb nitrate in a daily oral doseof 120mg/kg for 40-50 days (Secchiet al. Clinica chim. Acta 1970,27, 467). On the other hand, glutamate-dehydrogenase activity was reduced.Thesechangeswere the sameas those found in guinea-pigkidney tissuerenderedischaemic by occlusionof the renal artery, and were thought to indicatean impairmentof oxygen utilization and involvementof the mitochondria. Histologicalevidenceof renal tubular damagehas also beenobservedin other species.Chicksgiven I.0 or 0.5%Pb acetatein the diet for 3 weeksdeveloped necrosisof the epithelium of the renal proximal tubules,and inclusionbodies-similar to those seen in rats-were demonstratedby electron microscopy in the nuclei of cells from this part of the nephron (Simpsonet al. Am. J. vet. Res. 1970,31.515).Similar inclusionbodieswereseenin young dogsgiven 0.01% Pb acetatein the diet for 12 weeksfrom the age of 6 weeks,and foci of necrosiswere seenin the proxima1renal tubulesof theseanimals(Stoweet al. Archs Path. 1973, 95, 106).Cynomolgusmonkeysexposed virtually continuouslyfor 104weeksto Pb chlorobromidedispersedin particulate form in the atmosphere developeda nephropathycharacterizedby dilatation of the proximal convoluted tubulesand degeneration of their epithelial cells, which showed vacuolated cytoplasm, vesiculatednuclei, disruptedbordersand the presenceof intranuclearinclusions(Campbellet al. J. Am. vet. med. Ass. 1971,159, 1523). [P. GrasseBIBRA]
. . . . . MERCURY. . . . .
The secondarticle in this serieson the effects of mercury derivatives. Four children and two adults heavy metalson the mammaliankidney is concerned weregiven chloramphenicolinjectionsthat contained with the actionsof mercury (Hg). Merthiolate, a phenylmercuriccompoundusedextensively as an antisepticagent, in a concentration 1000 timesgreaterthan the correct level (Axton, Post-grad. Human experience med. J. 1972,48, 417). Five of the patients died, The nephrotoxic hazard from the useof Hg com- although three of them were treated with 2,3-dimerpoundseither medicinally,aspesticidesor in industry capto-1-propanol(BAL), and at autopsy the kidneys is well recognized,.but the advantagespossessed by wereswollenand necrosiswasapparentin the proximany Hg-basedcompoundsmakethe finding of ade- mal tubular cells. Each of these fatal caseshad quate substitutesa difficult task. The renal changes receivedbetween20 and 110mg Hg/kg or approxiproduced in experimental animals by the mately 70-330mg Merthiolate/kg. The oral LD,, of administrationof high dosesof Hg compoundsboth Merthiolate in rats is reported as 60 mg/kg. In at in the short and long term are reasonablywell estab- least two cases,gross albuminuria and glycosuria lished,but the picture in man is far lessclear. were observedand the one surviving child, who had Severalcasesof extensiverenal damageoccurring receiveda total doseof about 30 mg/kg, had traces in unexpectedcircumstances shedsomelight on the of albumin in its urine for sometime after cessation type of effect that may be inducedin man, at least of the chloramphenicoltreatment. by phenylmercuriccompounds,which are. perhaps Albuminuria and glycosuria have also been assomore commonly used medicinally than any other ciatedwith poisoningresultingfrom more prolonged
467
damage DNA has been exploited to provide a rapid plate test for mutagens, which involves measuring the inhibition of growth of parent and mutant strains (Kada et al. ibid 1974,26, 243; Slater et al. Cancer Res. 1971,31, 970).Longneckeret al. (ibid 1974,34, 1658) evaluated the test with a wide variety ,of mutagens,however, and concluded that it was not as sensitive as was first thought, especiallywith mutagensrequiring metabolic activation. Moreover, the observationthat for somerepair-deficientstrains the extent of killing by mutagensis unrelated to the number of induced mutations detracts somewhat from the value of the method (Hince & Neale,
luated most carefully in the light of the recognized limitations, but such tests, in combination with other short-term tests for mutagenicity including those involving mammaliancells in culture, would provide a most effective primary screen for detecting mutagens.This approach has been suggestedby Bridges(ibid 1974,26, 335) who hasoutlined a threetier systemof mutagenicitytesting,in which this primary screeningmay be followedby in viva testsusing whole mammalsand finally, in the light of the earlier resultsand potential levels of exposure,by experimentsdesignedto give somequantitative indication of the potential risk to man. Obviously, for a full Mutation Res. 1974, 22. 235). definition of the mutagenicpotential of a chemical In spite of the limitations discussedin this article, it is necessaryto determinemany more facts, such microbial mutagenicity testsare obviously the only as the mutagenconcentration at the site of action, meansavailablefor screeninglargenumbersof chemi- the active metabolic products and their rate of calsand drugs for mutagenicactivity, sincein terms absorption,metabolismand elimination. of speedand sensitivity they are vastly superior to in vivo testsin mammals.Clearly, both positive and [I. R. Rowland-BIBRA] negative results from microbial tests must be eva-
RENAL RESPONSE TO LEAD.. . . .
.
The three heavy metals,lead (Pb), mercury and readily than from the lysosomes,since the concadmium,are ingestedin smallamountswith many centration in both organelleswas virtually the same types of food and are currently the object of some 7 and 14daysafter treatment.Furthermore,previous concernto health authoritiesall over the world. Des- oral dosingw.ith stable Pb acetate(300 mg/kg/day) pite wide differencesin their absorptionand excretion for 7 consecutivedays had little effect on the lysosorates and in the pathologicalchangesthey may in- ma1 uptake of ‘03Pb, whereasthe mitochondrial duce, thesemetalspossess at least one common fea- uptake of the isotopewasreducedby 3040%. To someextent, thesebiochemicaldata have been ture, namelytheir ability to accumulatein the kidney and producerenal damage.The type of damagepro- reflected in the findings of an ultrastructural study duced by each element has distinctive features, by Murakami & Hirosawa(Nature, Lmd. 1973,245, although someeffectsare common to all three. This 153),who usedelectron-microscopic autoradiography article is the first of a short serieson thesemetals to demonstratethe presenceof “‘Pb within the mitoand is concernedspecificallywith the renal changes chondria of the tubular epithelial cellsof the kidney associatedwith Pb intoxication. 30 minutesafter administrationof an ip injectionconPb penetratesrapidly into the renal cells.Within taining this radioactive isotope. The mitochondriafrom Pb-treatedanimalsdisplay 1 hour of iv injection of .I00 g *l’Pb as Pb acetate into rats, the Pb was identified in all the fractions other typesof abnormality apart from thesePb incluof a renal homogenate(Castellino& Aloj, Br. J. ind. sions.Usually elongatedand rod-shaped,the mitoMed. 1969,26, 139).During the first 24 hours after chondria of the proximal renal tubulesbecomeoval a singleinjection, the amount of Pb in the nuclear or roundedand increasein volume (Goyer, Lab. Inand mitochondrial fractions increasedat the expense vest. 1968,19, 71).Cristaemay be vesicularbut more of that in the microsomalfraction, and throughout often they are shortenedand marginal,while matrical the subsequent 8 daysof the study the distribution re- granules become less homogeneousand tend to mainedrelatively stable,with considerablyhigherlevels clump. in the two former fractions than in the microMore significant,perhaps,is the informationon the somaland solublefractions.Washingwith 0.25M-SUCfunctional state of the mitochondria obtained by rose,aloneor containing0001M-ethylenediaminetetra- Goyer et al. (ibid 1968,19, 78) in a study designed acetic acid (EDTA), removed Pb lessreadily from to elucidatethe meaningof the observedmorphologithe mitochondrialthan from other fractions, indicat- cal changes.Mitochondria isolatedfrom the proximal ing somedegreeof stable binding. Further insight tubular cellsof rats fed a diet containing 1%Pb acerate into the distribution of Pb within the kidney cells tate for 6 weekswere found to have a decreased has been provided by Barltrop et al. (J. Lab. clin. of respirationand partially uncoupledoxidative phosMed. 1971,77, 705).After injectingradioactive ‘03Pb, phorylation. This functional defect was accentuated theseauthors subdividedthe original mitochondrial by in vitro ageingof the mitochondria at 0°C and fraction of the kidney homogenateinto mitochondrial was partially reversedby addition of EDTA. Moreand lysosomalfractionsand found that 2 hoursafter over, the mitochondrial outer membranes,where treatment the mitochondriacontained four timesas someof the respiratory enzymesare located, were much “‘Pb as the lysosomalfraction. It appears, ruptured. Further evidenceof an impairmentin the however,that Pb is lost from the mitochondriamore respiratory-enzyme‘activity of mitochondria from
468
Articles of general interest
thesePb-intoxicatedrats wasobtainedat a later stage of this work, when a significant reduction in cytochromesuag and h was demonstrated(Rhyne & Goyer, E.uplnzol.Path. 1971,14, 386). This mitochondrial defect may provide some explanation for the amino-aciduriaseenas a result of Pb poisoning,sincethe transport of amino acids in the proximal renal tubule is an active, energyrequiring process(Goyer et al. lot. cit.). The aminoaciduria occurring in Pb poisoninghasbeencharacterized as “generalized”and is usually accompanied by glycosuria and hyperphosphaturia(Chisolm, J. Pediat. 1962,60. 1). It was investigatedfurther by Goyer et al. (Proc. Sot. exp. Biol. Med. 1970,135, 767) who confirmed that in Pb-poisonedrats it is largely secondaryto impaired function of the renal tubules.They demonstrateda one- to threefoldelevation in the renal clearancesof mostamino acids; the increasein histidineclearancewasexceptionallylarge, but changesin glycine and valine clearanceswere slightand tyrosine clearanceactually decreased. This lossof amino acids is unlikely to be due entirely to the impairmentof tubular reabsorption,however,and the formation of metal-amino acid complexesmay be a contributory factor. This excessiveamino-aciduriadevelopswithin 3 weeksin rats fed 1% Pb acetate,and administration of Pb for longerperiodstendsto accentuatethe renal tubular damage.Feedingof 1 or 2% Pb acetate for 10-40weeksresultedin an increasein kidney weight and a decrease in the capacity of the kidney to synthesizeglucoseand metabolizepyruvate (Hirsch, Toxic. appl. Pharmac. 1973,25,84).Excretion of p-aminohippurate during the first 2 hours after administration was higher than that in controls. Feedingof a diet containing 200 ppm Pb or lessdid not affect these parameters.Histologicalexaminationrevealedvacuolar degenerationin the distal segmentof the proximal renal tubulesof rats subjected(either directly or, initially. via the milk of treateddams)to a diet contain-
ing 2% Pb acetate for the first 10 weeksof life. A 4% dietary level of Pb acetate caused,in addition, somecellular necrosis. The renal responseto Pb intoxication has been studiedlessintensively in other speciesthan in the rat, but the data available indicate that broadly speakingsimilar changesoccur. Levels of activity of lactate dehydrogenase and glucose-6-phosphate dehydrogenase were up to twice as high as normal in kidney homogenatesof guinea-pigsgiven Pb nitrate in a daily oral doseof 120mg/kg for 40-50 days (Secchiet al. Clinica chim. Acta 1970,27, 467). On the other hand, glutamate-dehydrogenase activity was reduced.Thesechangeswere the sameas those found in guinea-pigkidney tissuerenderedischaemic by occlusionof the renal artery, and were thought to indicatean impairmentof oxygen utilization and involvementof the mitochondria. Histologicalevidenceof renal tubular damagehas also beenobservedin other species.Chicksgiven I.0 or 0.5%Pb acetatein the diet for 3 weeksdeveloped necrosisof the epithelium of the renal proximal tubules,and inclusionbodies-similar to those seen in rats-were demonstratedby electron microscopy in the nuclei of cells from this part of the nephron (Simpsonet al. Am. J. vet. Res. 1970,31.515).Similar inclusionbodieswereseenin young dogsgiven 0.01% Pb acetatein the diet for 12 weeksfrom the age of 6 weeks,and foci of necrosiswere seenin the proxima1renal tubulesof theseanimals(Stoweet al. Archs Path. 1973, 95, 106).Cynomolgusmonkeysexposed virtually continuouslyfor 104weeksto Pb chlorobromidedispersedin particulate form in the atmosphere developeda nephropathycharacterizedby dilatation of the proximal convoluted tubulesand degeneration of their epithelial cells, which showed vacuolated cytoplasm, vesiculatednuclei, disruptedbordersand the presenceof intranuclearinclusions(Campbellet al. J. Am. vet. med. Ass. 1971,159, 1523). [P. GrasseBIBRA]
. . . . . MERCURY. . . . .
The secondarticle in this serieson the effects of mercury derivatives. Four children and two adults heavy metalson the mammaliankidney is concerned weregiven chloramphenicolinjectionsthat contained with the actionsof mercury (Hg). Merthiolate, a phenylmercuriccompoundusedextensively as an antisepticagent, in a concentration 1000 timesgreaterthan the correct level (Axton, Post-grad. Human experience med. J. 1972,48, 417). Five of the patients died, The nephrotoxic hazard from the useof Hg com- although three of them were treated with 2,3-dimerpoundseither medicinally,aspesticidesor in industry capto-1-propanol(BAL), and at autopsy the kidneys is well recognized,.but the advantagespossessed by wereswollenand necrosiswasapparentin the proximany Hg-basedcompoundsmakethe finding of ade- mal tubular cells. Each of these fatal caseshad quate substitutesa difficult task. The renal changes receivedbetween20 and 110mg Hg/kg or approxiproduced in experimental animals by the mately 70-330mg Merthiolate/kg. The oral LD,, of administrationof high dosesof Hg compoundsboth Merthiolate in rats is reported as 60 mg/kg. In at in the short and long term are reasonablywell estab- least two cases,gross albuminuria and glycosuria lished,but the picture in man is far lessclear. were observedand the one surviving child, who had Severalcasesof extensiverenal damageoccurring receiveda total doseof about 30 mg/kg, had traces in unexpectedcircumstances shedsomelight on the of albumin in its urine for sometime after cessation type of effect that may be inducedin man, at least of the chloramphenicoltreatment. by phenylmercuriccompounds,which are. perhaps Albuminuria and glycosuria have also been assomore commonly used medicinally than any other ciatedwith poisoningresultingfrom more prolonged