Alvin Segal

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

Abstract The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3, N 4 -bis(2-carboxyethyl)deoxycytidine (3, N 4 -BCEdCyd) (0.6% yield). 3-CEdCyd and 3, N 4 -BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3, N 4 -bis(2-carboxyethyl)cytosine (3, N 4 -BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d 27 ) tri-TMS derivative of 3-CEC. The structure of 3, N 4 -BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3, N 4 -BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3, N 4 -BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H 2 O at 37°C and pH 7.0–7.5.

In vitro transformation of C3H/10T1/2 and NIH/3T3 cells by acrylonitrile and acrylamide

Acrylonitrile (AN) and acrylamide (AM) are carcinogenic in a number of rodent organs and AN is a suspected human carcinogen. We sought to determine whether AN and/or AM could produce morphological transformation in vitro in C3H/10T1/2 and NIH/3T3 mouse fibroblast cells. Both AN and AM induced a dose-dependent cytotoxic effect in C3H/10T1/2 and NIH/3T3 cells and readily transformed both cell lines. Our conclusions are based on the appearance of cells exhibiting a transformed phenotype and growth in soft agar. AN and AM transformed NIH/3T3 cells to a greater extent than C3H/10T1/2 cells. This is the first reported transformation of cells in vitro by AM.

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA☆

Abstract 3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (∼3%). Phosphotriester formation was not detected.

Studies of the effects in vitro of β-propiolactone and β-propiolact[14C]one on whole mouse skin chromatin☆

Abstract Whole mouse skin chromatin was reacted with β-propiolactone (BPL) in vitro. The alkylated chromatin preparation was isolated essentially intact. The transition mid-point (Tm) of the alkylated chromatin preparation was reduced by 24°. its template activity for RNA synthesis was virtually abolished, its chromosomal RNA (cRNA) content was reduced and lysine-rich histones F1 and 1° could no longer be detected on polyacrylamide gels. DNA from this alkylated chromatin showed a similar decrease in Tm and template activity. The histones in alkylated chromatin stabilized DNA against heat denaturation to a similar extent compared to untreated chromatin. Mouse skin DNA alkylated with BPL had properties similar to those of DNA from alkylated chromatin. Buoyant density studies revealed that DNA isolated from alkylated chromatin exhibited peaks corresponding to alkylaled and normal DNA. Mouse skin DNA alkylated under identical conditions exhibited a single peak for alkylated DNA. Thus portions of DNA in mouse skin chromatin appeeared to be protected against alkylation in vitro with BPL. This was confirmed in studies using β-propiolact[14C]one (BPL-14C). The alkylation reactions were repeated using reduced quantities of BPL-14C. DNA isolated from BPL-14C-alkylated whole mouse skin chromatin contained 29 per cent of the bound radioactivity found in mouse liver DNA alkylated under identical conditions. Acidic proteins and histones as well as DNA were shown to bind BPL-14C. Among the histone classes, F1 and 1° contained the greatest amount of bound BPL-14C.

ALTERATION OF HISTONES FROM MOUSE EPIDERMAL CELLS AFTER INCUBATION WITH ELASTASE AND HYALURONIDASE

Chromatin was isolated from whole mouse skin, mouse epidermal cells and mouse liver by standard procedures used for isolation of chromatin from other mammalian tissues. Chromatin from whole mouse skin or from mouse epidermal cells had not been isolated or characterized earlier. For the preparation of chromatin from mouse epidermal cells, the latter was separated from dermis by incubation for 30 min at 37°C in a solution containing the enzymes elastase and hyaluronidase. The relative proportions of the chromatin components, the T m and the ultraviolet absorption spectrum were all similar to that of chromatin from whole mouse skin which was not treated with enzymes and to other mammalian chromatin preparations. Electrophoresis of the histones from epidermal chromatin in polyacrylamide gels revealed the absence of histones F1, F3 and F2a2 and the appearance of a new band. Histones isolated from chromatin prepared from the whole mouse skin had a gel electrophoresis pattern virtually identical with histones isolated from mouse liver chromatin and to reported histone patterns from other mammalian tissues. The alterations in mouse epidermal histones are similar to reported changes in histones from calf thymus nucleohistone previously subjected to incubation at various temperatures. The enzymatic incubation technique can therefore not be used as a method of isolating unaltered mouse epidermal chromatin. The findings illustrate that very subtle chemical alterations can be induced by usual methods of tissue preparation and that these changes can only be detected by highly sensitive analytical techniques.

Phosphate diester formation following reaction of β-propiolactone with thymidine-5′-monophosphoric acid☆

Abstract The carcinogen β-propiolactone (BPL) was reacted with thymidine-5′-monophosphoric acid (dThd5′P) at pH 1.8 and various temperatures in order to determine whether BPL forms phosphodiesters with dThd5′P. The acidic conditions were chosen in an attempt to avoid complicating ring alkylations. Paper chromatographic analyses of the reaction products revealed the presence of unreacted dThd5′P and a second ultraviolet absorbing compound designated Compound F2. F2 and dThd5′P had identical UV spectra at pH 1 and 13. The major component of the reaction mixture was unreacted dThd5′P. F2 represented 10% of the ultraviolet absorbing compounds extracted from paper chromatograms following reactions at 37, 50, 63 and 76°C and 5% at 24°C. On the basis of ultraviolet spectra, treatment with snake venom phosphodiesterase and alkaline phosphatase (each at pH 7.2 and 9.0), labeling experiments with β-[ 14 C]propiolactone and hydrolyses at pH 1, 7, 9, 11 and 13 at 37°C for 18 h (no detectable effects) the new phosphodiester, 5′-O-(2-carboxyethyl)phosphonothymidine was assigned to F2. When BPL was reacted with dThd5′P at pH 6.6–7.0 at 37°C, 2 new UV absorbing compounds were observed on paper chromatograms in addition to dThd5′P and F2, and were designated Compounds F3 and F4. UV spectra of F3 and F4 were identical to dThd5′P at pH 1 and 13. dThd5′P represented 36% of the ultraviolet absorbing compounds extracted from paper chromatograms, the remainder being F2 (36%), F3 (24%) and F4 (4%). Treatment of F3 with snake venom phosphodiesterase at both pH 7.2 and 9.0 resulted in complete conversion to dThd5′P. F3 is thus a phosphodiester. Hydrolysis of F3 at pH 13 and 11 (37°C and 18 h) resulted in complete conversion of F3 to F2 and a trace amount of dThd5′P while F3 was unreactive at pH 9, 7 and 1. 3′-O-Acetylthymidine was completely hydrolyzed to thymidine at pH 13, 11 and 9 and partially (65%) hydrolyzed at pH 1. A 3′-derivative of dThd5′P (ether or ester) was thus ruled out as a structure for F3. After considering a number of structures, a tentative assignment of a structure containing a 6-membered ring phosphate ester-acyl phosphate derivative was assigned to F3 based on the evidence presented and a positive acyl phosphate test for F3. When the reaction between BPL and dThd5′P was conducted at pH 7.0–7.5, 7.5–8.0 and 8.0–8.5 the results were similar to the reaction conducted at pH 6.6–7.0. No ring alkylation was detected. The significance of these findings in terms of possible effects of BPL on messenger RNA activity, excision-repair of DNA, DNA synthesis and possible alterations in gene expression following reaction of BPL with phosphorylated histones and nonhistone chromosomal proteins is discussed.

In vitro acylation of the ϵ-amino group of l-lysine in calf thymus histones by the carcinogen, β-propiolactone

Abstract We had previously reported that the carcinogen, β-propiolactone (BPL) reacted in vitro with histones in whole mouse skin chromatin and that among the histone classes BPL was preferentially bound to the lysine-rich histones H1 and H1°. In order to determine if in vitro reaction of BPL with calf thymus histones resulted in binding of BPL to l -lysine, we synthesized the model compounds ϵ- N -(3-hydroxypropionyl)lysine (HPL) and ϵ- N -(2-carboxyethyl)lysine (CEL) from BPL and l -lysine. The α-amino group of l -lysine was protected from reaction with BPL by the formation of a copper chelate. Structures were assigned on the basis of infrared spectra, pKa values and chemical analyses. BPL was reacted in vitro with calf thymus histones and the BPL-reacted calf thymus histones and control calf thymus histones were digested with trypsin followed by pronase. The respective digests were each chromatographed on a column of AA-15 cation-exchange resin. The elution profiles of the two digests were very similar except for the appearance of a new ninhydrin-positive peak (NNPP) in the eluate of the trypsin-pronase digest of BPL-reacted calf thymus histones. When compounds HPL and CEL were added to the trypsin-pronase digest of control calf thymus histones and the mixture chromatographed on AA-15, both compounds were resolved from the other peptide (or amino acid) peaks. HPL was eluted in the same fractions as NNPP, HPL and NNPP exhibited identical R F values on silica gel TLC with acidic, alkaline and neutral solvents. CEL was not identified as a product of the reaction between BPL and calf thymus histones.

Inducibility of thymic lymphomas in female Hsd: (ICR)BR mice following subcutaneous injection of ethyl- and isopropyl methanesulfonate

Isopropyl methanesulfonate (IMS), a direct-acting SN1 alkylating agent, induced thymic lymphomas in 29 of 30 female Hsd: (ICR)BR mice by the 139th day following once a week subcutaneous injections at a dose of 10 mumol/mouse. No such neoplasms were observed at the 450th day following 52 once a week subcutaneous injections (20 mumol/mouse) of the borderline SN1/SN2 alkylating agent ethyl methanesulfonate (EMS). Local sarcomas were observed in 2 and pulmonary adenomas in 20 of the 30 EMS-treated mice, 70% of which were alive at the 450th day. Neoplasms were not observed at local or at distant sites other than the thymus gland following treatment with IMS. The results suggest a sensitivity of hemopoietic tissue to the types of DNA lesions induced by IMS.

Rapid Induction of Thymic Lymphomas by Isopropyl Methanesulfonate: A Preliminary Report

Abstract The direct-acting SN1 alkylating agent isopropyl methanesulfonate (IMS) was carcinogenic by subcutaneous injection in female Hsd:(ICR)BR mice, causing thymic lymphoid neoplasms within 7 months in at least 20 of 32 treated mice. No such neoplasms were observed in mice treated with the direct-acting SN2 methyl homolog, methyl methanesulfonate (MMS). Both the IMS-treated mice and the MMS-treated mice initially received 20 μmole of the respective compounds by sc injection once weekly; however, because of toxic effects the dose of IMS was reduced to 10 μmole per injection on the 63rd day and further reduced to 5 μmole per injection on the 120th day, after which this dose was maintained until the 202nd day when the last surviving IMS-treated mouse became moribund and was sacrificed. In 2 of the MMS-treated mice, 93% of which were alive at 288 days, tumors were observed at the site of injection, one being a papilloma and the other a subcutaneous sarcoma. IMS has not previously been implicated as a carcinogen, to our knowledge. Its induction of thymic lymphomas may conceivably be related to its ability to alklyate exocyclic oxygen atoms in the DNA of hemopoietic cells.

A New Personal Monitoring Device for the Detection of β-Propiolactone and Other Alkylating Agents

A personal monitoring badge has been developed for the detection of the direct-acting, alkylating carcinogen beta-propiolactone at atmospheric concentrations as low as 6 ppb for 24-hour and 0.6 ppm for 0.25-hour exposure. The method employs the trapping reagent p-nitrobenzyl pyridine (p-NBP) absorbed on a cellulose thin-layer chromatography (TLC) strip. Deoxyguanosine can be used in place of p-NBP, but its lower limit of detection is 60 ppb for 24-hour exposure. The authors also obtained positive results with the carcinogens bis (chloromethyl) ether, chloromethyl methyl ether, diepoxybutane, dimethylcarbamoyl chloride, ethyleneimine, and glycidaldehyde. In practice, the TLC strip is positioned in a filmbadge holder. The TLC strip monitoring badges are easy to prepare; they should encounter no resistance of personnel to their use since they are not cumbersome. Monitoring at the end of an exposure is simple and requires no expensive equipment or specialized personnel.