The absorption, metabolism and tissue distribution of di(2-ethylhexyl)phthalate in rats

Rats given a single oral dose of [¹⁴C] di(2-ethylhexyl) phthalate [¹⁴C] (DEHP) excreted 42% and 57% of the dose in the urine and faeces respectively in 7 days. A significant proportion (14%) of the dose is excreted in bile. Rats fed 1000 ppm DEHP in the diet for 7 days prior to dosing with [¹⁴C] DEHP excreted 57% and 38% in the urine and faeces respectively in 4 days.When fed continuously to rats at dietary concentrations of 1000 and 5000 ppm, the amount of the ester in liver and abdominal fat rapidly attains a steady-state concentration and there is no evidence of accumulation. When returned to a normal diet, the radioactivity in the liver declined with a half life of 1–2 days while that in fat declined rather more slowly to give a half life of 3–5 days. The relative liver weight increased to a level 50% above normal in rats receiving 5000 ppm DEHP and returned to normal within 1 week after being returned to normal diet.When administered intravenously DEHP is preferentially localised in lung, liver and spleen from where it is eliminated with a half-life of 1–2 days.The hexobarbital sleeping time was reduced by 30–40% in rats following repeated oral administration of DEHP; when the ester was administered intravenously sleeping time was increased by approx. 40%.DEHP is extensively metabolised after oral administration, the principal metabolites being identified as the acid, alcohol and ketone resulting from ω- and (ω-1)-oxidation of mono(2-ethylhexyl) phthalate (MEHP). DEHP is rapidly hydrolysed to the half-ester by pancreatic lipase.     

Elimination,distribution and metabolism of di-(2-ethylhexyl)adipate (deha) in rats

The excretion, retention, distribution and metabolism of di-(2-ethylhexyl)adipate (DEHA) have been studied in the rat.After oral administration of [¹⁴C]DEHA, almost all the dose was excreted within 48 h, predominantly in the urine and as respiratory carbon dioxide. The faecal excretion was low. There was no evidence of the accumulation of radioactivity in any organs or tissues. Adipic acid (AA) was found to be the main urinary metabolite; it was also detected in the digestive tract, blood and liver.In vitro, DEHA was hydrolyzed at a significant rate by tissue preparations prepared from liver, pancreas and small intestine of the rat.These results suggest that orally administered DEHA is rapidly hydrolyzed in the body to form AA without any accumulation of mono-(2-ethylhexyl)adipate (MEHA).     

Di-(2-ethylhexyl)adipate: absorption, autoradiographic distribution and elimination in mice and rats

Whole-body autoradiography was used to study the tissue distribution of the plasticizer di-(2-ethylhexyl) adipate (DEHA), labelled in the acid [carbonyl-14C] or alcohol [2-ethylhexyl-1-14C]moiety, after iv or ig administration to male mice and rats and pregnant mice. With both DEHA preparations, during the first 24 hr after administration high levels of radioactivity were observed particularly in the body fat, liver and kidneys (after iv and ig administration) and in the intestinal contents (after ig administration) of both species. After administration of [carbonyl-14C]DEHA, radioactivity was also registered in the adrenal cortex, corpora lutea of the ovary, bone marrow, forestomach mucosa, salivary glands and Harder's gland in both species. [2-ethylhexyl-1-14C]DEHA derived radioactivity was found in the bronchi in male mice. Radioactivity was observed in the foetal liver, intestine and bone marrow during the first 24 hr after iv or ig administration of [carbonyl-14C]DEHA to pregnant mice. There was very little accumulation of [2-ethylhexyl-1-14C]DEHA in the mouse foetus but some was found in the urinary bladder, liver and intestinal contents as well as in the amniotic fluid. In an absorption/elimination study in rats of doses of 25 microCi/kg body weight of [14C]DEHA administered ig, dissolved in corn oil or dimethylsulphoxide, blood levels of radioactivity increased somewhat faster and were two or three times higher when DMSO was the vehicle indicating poor absorption of DEHA from the corn oil solution which more accurately reflects human contact with DEHA. Little radioactivity from [carbonyl-14C]DEHA was recovered in the bile, whereas [2-ethylhexyl-1-14C]DEHA was excreted in the bile in significant amounts particularly when DMSO was the vehicle. There was evidence of enterohepatic circulation of DEHA. Radioactivity was also excreted in the urine. As shown by autoradiograms obtained 4 days after the administration of [14C]DEHA there was no retention of DEHA and/or its metabolites in the tissues of mice.     

Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate

Di(2-ethylhexyl) phthalate (DEHP) is a well-known testicular toxicant inducing adverse effects in androgen responsive tissues. Therefore, di(2-ethylhexyl) adipate (DEHA) is currently being evaluated as a potential substitute for DEHP. Similarities in structure and metabolism of DEHP and DEHA have led to the hypothesis that DEHA can modulate the effects of DEHP. Wistar rats were gavaged with either vehicle, DEHP (300 or 750 mg/kg bw/day) or DEHP (750 mg/kg bw/day) in combination with DEHA (400 mg/kg bw/day) from gestation day (GD) 7 to postnatal day (PND) 17.

Decreased anogenital distance (AGD) and retention of nipples in male offspring were found in all three exposed groups. Dosed males exhibited decreased weights of ventral prostate and m. levator ani/bulbocavernosus. Histopathological investigations revealed alterations in testis morphology in both juvenile and adult animals. The litter size was decreased and postnatal mortality was increased in the combination group only, which is likely a combined effect of DEHP and DEHA. However, no combination effect was seen with respect to antiandrogenic effects, as males receiving DEHP in combination with DEHA did not exhibit more pronounced effects in the reproductive system than males receiving DEHP alone.     

Studies on the effects of orally administered di-(2-ethylhexyl) phthalate in the ferret

A target-organ study of the effects of the phthalate ester di-(2-ethylhexyl) phthalate (DEHP) has been conducted in mature male albino ferrets. DEHP treatment caused a loss of body weight when administered as a 1% (w/w) diet for 14 months. Additionally, marked liver enlargement with associated morphological and biochemical changes was observed. These changes consisted of liver cell enlargement, lysosomal changes, dilatation of the endoplasmic reticulum and the depression of a number of marker enzyme activities. The only other tissue observed to be affected by DEHP treatment was the testes where histological evidence of tissue damage was observed in some animals.Studies on the metabolism of [¹⁴C]DEHP in the ferret indicated that the diester was metabolised to derivatives of mono-(2-ethylhexyl) phthalate which were excreted in the urine both unconjugated and as glucuronides.The results obtained have been compared with previous studies in the rat and it is concluded that DEHP is hepatotoxic in both species.     

Toxicity of di(2-ethylhexyl)phthalate (DEHP) and di(2-ethylhexyl)adipate (DEHA) under conditions of renal dysfunction induced with folic acid in rats: enhancement of male reproductive toxicity of DEHP is associated with an increase of the mono-derivative

F344 male rats were given five consecutive weekly subcutaneous injections of folic acid for induction of chronic renal dysfunction and then di(2-ethylhexyl)phthalate (DEHP) or di(2-ethylhexyl)adipate (DEHA) in the diet at a concentration of 0, 6000 or 25,000 ppm for 4 weeks in order to investigate whether male reproductive toxicity of the two chemicals might be enhanced under conditions of renal disease. Control animals also received DEHP or DEHA in the same manner but without folic acid pretreatment. Decreased testicular weights, seminiferous atrophy with vacuolization of sertoli cells and diminished sperm counts were more prominent in rats given folic acid and then 25,000 ppm DEHP as compared to those exposed to DEHP alone. No such reproductive toxicity was evident in rats given 6000 ppm DEHP or either dose of DEHA. An increased concentration of the mono-derivative of DEHP (mono(2-ethylhexyl)phthalate, MEHP) in the blood, testis and urine was considered relevant to the enhanced reproductive toxicity observed with DEHP.     

Biochemical studies on phthalic esters I. Elimination, distribution and metabolism of di-(2-ethylhexyl)phthalate in rats.

Elimination, distribution and metabolism of di-(2-ethylhexyl)phthalate (DEHP) were studied in the rat by the tracer technique. About 80% of the dose was excreted in the urine and faeces in 5 to 7 days following intravenous or oral administration. Excretion in the urine was generally slightly greater than that in the faeces. After intravenous administration of [14C] DEHP the radioactivity was preferentially localized in the liver for a short period. Delayed excretion of DEHP was observed in particular in adipose tissue. After oral dosing no significant retention was found in organs and tissues. Radioactivity measurements showed that affinity was lowest for testicles and brain regardless of whether [14C] DEHP was administered orally or intravenously. Orally ingested DEHP was excreted unchanged in the faeces and four major metabolites were detected in the urine.     

Studies on the hepatic effects of orally administered di-)2-ethylhexyl) phthalate in the rat.

A sequential study of hepatic effects was conducted in young male Wistar albino rats following daily oral intubation of di-(2-ethylhexyl) phthalate (DEHP) at a dose of 2000 mg/kg for 21 days. The relative liver weights of the animals increased progressively with the duration of the treatment. Alcohol dehydrogenase activity and microsomal protein and cytochrome P-450 contents showed a marked initial increase followed by a reversal as the treatment progressed. In contrast to this biphasic response, the activities of microsomal glucose-6-phosphatase, aniline 4-hydroxylase, and mitochondrial succinate dehydrogenase activity were significantly depressed, as observed both biochemically and histochemically, throughout the period of exposure. Parallel electronmicroscopic studies revealed a progressive dilatation of the smooth and rough endoplasmic reticulum, mitochondrial swelling and an increase in microbodies. Investigations carried out on the metabolic fate of [¹⁴C]DEHP in animals prior to and during the course of DEHP treatment showed no significant differences in the excretion pattern of radioactivity. Furthermore, there was no evidence indicating the storage of phthalate residues in the liver. Studies on the comparative effects of phthalic acid, 2-ethylhexanol, and mono-(2-ethylhexyl)phthalate (MEHP) orally administered at dose levels equimolar to DEHP for 7 days showed that the biochemical and ultrastructural changes in the hepatic endoplasmic reticulum and mitochondria in DEHP pretreatment were substantially reproducible by the administration of MEHP. Additionally, it was found that 2-ethylhexanol treatment led to an increase in the number of microbodies. The results indicate that the partial hydrolysis of DEHP to the monoester (MEHP) is the degradative step which determines the hepatic changes produced by DEHP.     

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.     

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro

[¹⁴C]di-n-octyltin dichloride ([¹⁴C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = μanol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNAbinding ability is about 30 000 times lower than the corresponding value for the strong carcinogen, aflatoxin b₁. In addition, [¹⁴C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.     

Disposition of orally administered di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate in the rat

The dispositon of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) was studied in the rat. Three hours after a single oral dose of DEHP (2.8 g/kg), plasma concentrations of 8.8±1.7 g/ml DEHP and 63.2±8.7 g/ml MEHP were reached. MEHP levels declined with a half-life of 5.2±0.5 h. The ratio of the area under the plasma concentration-time curve of MEHP to that of DEHP was 16.1±6.1. When ¹⁴CDEHP was administered, 19.3±3.3% of the radioactivity was excreted in the urine within 72 h, the rest being excreted in the faeces. The urinary excretion rate of total radioactivity declined with a half-life of 7.9±0.5 h. Single administration of MEHP (0.4 g/kg) resulted in plasma concentrations of 84.1±14.9 g/ml 3 h after dosing; the half-life of MEHP was 5.5±1.1 h. Multiple dosing with DEHP (2.8 g/kg/day) for 7 consecutive days produced no accumulation of DEHP or MEHP in plasma.     

Tissue distribution and excretion of tri-(2-ethylhexyl)trimellitate in rats

The disposition kinetics of tri-(2-ethylhexyl)trimellitate (TEHTM), a new plasticizer for polyvinyl chloride (PVC) plastic, was studied in rats following intravenous administration of [14C-carbonyl]tri-(2-ethylhexyl)trimellitate using an oil in water emulsion as the vehicle. The distribution half-life, elimination half-life, and clearance values estimated from the plasma concentration of radioactivity data obtained following iv administration of 10.5 mg/kg of TEHTM (59.9 muCi/kg), were 46.2 min, 5.34 d, and 40.5 ml/kg X h, respectively. Following iv dosage of 15.6 mg/kg of TEHTM (28.0 muCi/kg), significant accumulation of radioactivity was found in the liver, lungs, and spleen, with liver accounting for 72% of the administered dosage at 24 h. Excretion of TEHTM and its biotransformation products was slow, with 21.3% of the administered radioactivity found in the feces and 2.8% in the urine during the 14-d collection period. Biliary excretion seems to be the major route of elimination of TEHTM. The pharmacokinetic data gathered in the present investigation are compared to di-(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for PVC.     

Toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in rats

The toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP, was investigated in Sprague-Dawley rats orally treated with a single dose of 14C-DEHP. Urinary excretion of total 14C-DEHP and of its metabolites was followed by liquid scintillation counting (LSC). Concentrations of DEHP and MEHP were determined 6, 24, and 48 h after treatment in rat serum and 6, 12, 24, and 48 h after treatment in urine by high-performance liquid chromatography (HPLC). After 24 h, peak concentrations of MEHP in both urine and serum were observed in animals treated with 40, 200, or 1000 mg DEHP/kg. HPLC showed that general toxicokinetic parameters, such as Tmax (h), Cmax (microg/ml), Ke (1/h), and AUC (microg-h/ml/) were greater for MEHP than DEHP in both urine and serum. In contrast, the half-lives (t1/2 [h]) of DEHP were greater than those of MEHP. The AUC ratios between DEHP and MEHP were relatively smaller in serum than in urine, suggesting the important role of urinary DEHP data for exposure assessment of DEHP. The toxicokinetic relationship between DEHP and MEHP in rats suggests that DEHP exposure assessment should be based on DEHP and MEHP in urine and serum for risk assessment applications.     

Subcellular distribution of di-(2-ethylhexyl)phthalate in rat testis

Subcellular distribution of di-(2-ethylhexyl)phthalate (DEHP) in the testis was studied by single oral administration of [3,4,5,6-(3)H]-phthalic acid di-(2-ethylhexyl) ester (DEHP-3H) or phthalic acid di-(2-ethyl[1-(3)H]hexyl) ester (3H-DEHP) to 8-week-old male rats. Autoradiographs and electron microscopic autoradiographs were prepared from the testis, liver and kidney at 6 and 24 hr after administration and distribution of radioactive materials in the tissues were observed. In the autoradiographic specimen at 6 hr after administration of DEHP 3H-labeled at phthalic acid moiety (DEHP-3H), many grains were observed in the testis, mainly at the basal area of seminiferous tubules at the stages IX to I of the spermatogenic cycle. Electron microscopic autoradiographs taken at the same time revealed that localization of grains were in the smooth-surfaced endoplasmic reticulum and mitochondria of Sertoli cells. A few grains were also present at the Golgi apparatus and lysosome of Sertoli cells, and at the interfaces between the Sertoli cells or between Sertoli cells and spermatocytes, and in the cytoplasm of spermatocytes. Autoradiographs of the liver revealed grains in the centrilobular hepatocytes, localized at mitochondria, rough-surfaced endoplasmic reticulum and peroxisomes. In the kidney, the radioactivity was localized at the brush border of the tubular cells in the pars recta of proximal tubules. In the 24-hr specimen, the grain density in the seminiferous tubules obviously decreased. On the other hand, by autoradiography with DEHP 3H-labeled at the alcohol (3H-DEHP), only a few grains were observed in autoradiographs of the testes at 6 hr after administration. No grains were noted in autoradiographs of the liver and kidney with 3H-DEHP. The results showed that the phthalic acid ester was splitted rapidly in the body and only the phthalic acid moiety distributed into the cells.     

Peroxisome proliferation due to di (2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexanoic acid

The dose-response relationships for peroxisome proliferation due to Di (2-ethylhexyl) adipate (DEHA), 2-ethylhexanol (EH), 2-ethylhexanoic acid (EHA) have been investigated in rats and mice. Linear dose-response relationships were observed for induction of cyanide-insensitive palmitoyl CoA oxidation (PCO), used as a enzyme marker of peroxisome proliferation, by DEHA, EH and EHA in both species. Relative liver weights were also increased in a dose related manner. On a molar basis, DEHA was twice as potent as EH or EHA which were equipotent and PCO was stimulated to a greater extent in male mice than in rats or female mice. At doses above 8 mmol/kg/day, EH was toxic to rats (both sexes) and similarly EHA at 13.5 mmol/kg/day lead to the death of female rats. In a attempt to explain the species difference in carcinogenicity of DEHA previously reported, we also used Fischer 344 rats and B6C3F1 mice. DEHA administration (2.5 g/kg/day) to Fischer 344 rats and B6C3F1 mice lead to toxicity in female rats. Relative liver weights were increased in a dose related fashion by DEHA administration to both rats and mice, PCO but not catalase was markedly increased (up to 15 fold in male rats). Light microscopy examination indicated some glycogen loss, a dose related hypertrophy and increased eosinophilia in both rats and mice. Electron microscopy confirmed peroxisome proliferation accompanied by a marked reduction of lipid in the centrilobular hepatocytes. These data suggest EHA to be the proximate peroxisome proliferator derived from DEHA. These data indicate a higher sensitivity for Fischer 344 rats than B6C3F1 mice to hepatic peroxisome proliferation due to DEHA and ratio of PCO activity and catalase activity data suggest that more hydrogen peroxide (H₂O₂) could escape from peroxisomes in male Fischer 344 rats than B6C3F1 mice. These data obtained with B6C3F1 mice and Fischer 344 rats are not agreement with the carcinogenicity bioassays previously reported showing an incidence of hepatic tumours only in B6C3F1 mice.     

Excretion and metabolism of di(2-ethylhexyl)-phthalate in man

1. Di-(2-ethylhexyl)phthalate (DEHP) taken orally by two volunteers (30 mg each) was excreted in the urine to the extent of 11 and 15% of dose.

2. After enzymic hydrolysis the urinary metabolites (derivatives of mono (2-ethylhexyl) phthalate) were methylated and identified by g.l.c.-mass spectrometry (C.I.), and the quantitative distribution of conjugated and free metabolites determined.

3. DEHP taken by the same volunteers over a period of four days at doses of 10 mg daily gave no evidence of accumulation; 15 and 25% of the total dose was recovered in the urine.     

The influence of di(2-ethylhexyl)phthalate on protein turnover in rat liver

Treatment of rats with the plasticizer di(2-ethylhexyl)phthalate increases liver weight and leads to proliferation of mitochondria and peroxisomes. Using in vivo labelling with [3H]leucine, an increased rate of incorporation into the total protein of mitochondria and microsomes was observed. The half-lives of proteins in subcellular fractions were determined using [35S]methionine labelling. The half-lives for the total protein of mitochondria, microsomes, and supernatant were increased from 6 to 25 days, from 3.5 to 5.5 days and from 2.5 to 5 days upon treatment with phthalate esters. Experiments with [14C]guanidino-L-arginine indicated that some reutilization of [35S]methionine occurred, but this did not influence the results substantially. It appears that phthalate esters increase protein synthesis and decrease protein breakdown, the former effect being of greater importance.     

In vitro metabolism of di(2-ethylhexyl) phthalate (DEHP) by various tissues and cytochrome P450s of human and rat

In vitro metabolism of DEHP by subcellular fractions of human brain, intestine, kidney, liver, lung, skin, testis, rat liver and recombinant CYP isoforms of human and rat was investigated using LC–MS/MS. DEHP was rapidly hydrolyzed to mono(2-ethylhexyl) phthalate (MEHP) in 12 microsomal/cytosolic fractions of selected 7 human organs and rat liver but not in microsomal fractions of human brain and human female skin. MEHP was metabolized to CYP-mediated oxidative and dealkylated metabolites in human and rat liver and at a lower rate in human intestine. Measurable amounts of mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-Oxo MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5-carboxy MEPP), mono(2-carboxymethyl-hexyl) phthalate (2-carboxy MMHP) and phthalic acid (PA) were formed by human liver fractions. Human CYP2C9¹1, CYP2C19 and rat CYP2C6 were the major CYP isoforms producing 5-OH MEHP and 5-Oxo MEHP metabolites; however, only human CYP2C9¹1 and 2C9¹2 produced 5-carboxy MEPP from MEHP. Additionally, human CYP3A4 and rat CYP3A2 were the primary enzymes for PA production via heteroatom dealkylation of MEHP. Percent total normalized rates (%TNR) by CYP2C9¹1 in human liver microsomes (HLM) were 94%, 98% and 100%, respectively, for 5-OH MEHP, 5-Oxo MEHP, 5-carboxy MEPP, and 76% for PA production by CYP3A4.     

The metabolism and binding of ( 14 C)mycophenolic acid in the rat

Seventeen h after the intraperitoneal administration of 33 mg/kg of [¹⁴C]mycophenolic acid to rats, radioactivity was bound to the tissues of the intestines, bladder, stomach, kidney, liver and lung in decreasing order; no binding to spleen tissue was observed. In vitro incubations of the agent with macromolecules resulted in the binding of radioactivity to salmon sperm DNA and to bovine plasma albumin, the extent of binding being increased and decreased, respectively, in the presence of a rat liver microsomal system. The binding was apparently covalent since repeated purification procedures failed to release the bound radioactivity; heating of [¹⁴C]mycophenolic acid bound-DNA in n hydrochloric acid at 100° for 2 h caused the release of the bound radioactivity. Under the conditions described, 43% of the administered radioactivity was excreted in the urine (33%) and faeces (10%); the urine contained free mycophenolic acid (13%), mycophenolic acid glucosiduronate (17%) and an uncharacterized metabolite (3%).     

Instability of (ara-C)DNA under alkaline conditions

We employed both [5-³H]ara-C and [2-¹⁴C]ara-C labeled L1210 DNA for analysis following exposure to alkali under various conditions. The results demonstrated that the tritium label on C₅ of ara-C molecules incorporated in DNA was exchanged with water under alkaline conditions and, therefore, radioactivity was subsequently detectable in the acid-soluble fraction. The [¹⁴C]ara-C labeled DNA, however, was not susceptible to loss of radioactivity by this mechanism, and the appearance of this isotope in the acid-soluble fraction required degradation of the DNA strand or pyrimidine ring. Our results indicated that the [¹⁴C]ara-C labeled DNA was degraded by alkali, suggesting structural instability of this abnormal nucleic acid. These findings provide useful technical information on the purification of (ara-C)DNA labeled with different isotopes.