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Chapter 10 Modified Nucleosides as Biochemical Markers of Asbestos Exposure and Aids*
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CHAPTER 10 MODIFIED NUCLEOSIDES AS BIOCHEMICAL MARKERS OF ASBESTOS EXPOSURE AND AIDS* OPENDRA K. WARMAl, P h . D . and ALF FISCHBEINZ, M . D . ' L a b o r a t o r y o f M o l e c u l a r B i o l o g y , AMC C a n c e r R e s e a r c h C e n t e r , 1600 P i e r c e S t r e e t , Denver, Colorado
2 D i v i s i o n o f E n v i r o n m e n t a l and O c c u p a t i o n a l W e d i c i n e , Mount S i n a i S c h o o l o f M e d i c i n e o f t h e C i t y U n i v e r s i t y o f New Y o r k , One G u s t a v e L . Levy P l a c e , New Y o r k , New Y o r k
TABLE OF CONTENTS
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . C321 10.2 Results and Discussion . . . . . . . . . . . . . . . . . C324 10.2.1 Asbestos Exposure and Modified Nucleosides . . . . C324 10.2.2 Urinary Excretion of Modified Nucleosides in Patients With Various Manifestations of Infection With H I V . . . . . . . . . . . . . . . . . . C331 10.3 References . . . . . . . . . . . . . . . . . . . . . . C337
10.1 INTRODUCTION Cancer p a t i e n t s and tumor-beari ng animals excrete in t h e i r urine increased amounts of modified purines and pyrimidines ( r e f s . 1-6). These modified nucleosides, synthesized a t the macromolecular level ( r e f . 7 ) , are primarily constituents of t R N A and t o a l e s s e r extent of other RNAs. When RNA i s catabolized, most of these modified nucleosides cannot be r e u t i l i z e d ; consequently they a r e excreted. Pioneering s t u d i e s o f Borek e t a l . ( r e f . 8) have suggested t h a t excretion of elevated amounts of modified nucl eosides in tumor-beari ng animal s resul t s from increased t R N A turnover r a t h e r than from c e l l death. The molecular mechanisms of elevated excretion a r e unclear. Extracts of neopl a s t i c t i s s u e s have aberrant t R N A methyltransferases ( r e f . 7 ) and i t has been suggested t h a t the high turnover of t R N A i s due t o rapid degradation of aberrantly modified tRNAs ( r e f s . 7 , 8 ) . * T h i s work was supported by USPHS Grants HL-32432, HD-20612 OH-02122, NIEHS Center Grant E S 00928, CDC Grant OH-02122 and a g i f t t o t h e AMC Cancer Research Center from G e r a l d M . Q u i a t .
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More recent studies indicate t h a t catabolism of other RNA species and altered RNA metabolism of host tissue (refs. 5, 9 ) may also contribute t o elevated urinary levels of modified nwleosides. The urinary excretion of modified nucleosides and j-AIBA (degradation product of thymine) expressed re1 a t i ve t o creati n i ne i s remarkably constant for normal 18-60 year o l d subjects (refs. 1, 10). Infants and children excrete lower levels o f modified nucleosides compared t o adults (ref. 2 ) . Patients suffering from non-cancerous diseases do n o t excrete significantly elevated levels of modified nucleosides (ref. 11), however, elevated excretion i s observed i n some patients w i t h acute hepatitis (ref. l l ) , g o u t and psoriasis (ref. 12). Children w i t h acute infections do n o t excrete significantly elevated levels of modified nucleosides when compared t o healthy children (ref. 13). S l i g h t elevations i n u r i n a r y modified nucleoside excretion from subjects w i t h bacterial pneumonia and significant elevations from some subjects w i t h urinary tract infections have been noticed ( r e f . 143. Therefore, urinary excretion of modified nucleosides should be interpreted w i t h caution i n subjects w i t h syndromes t h a t interfere w i t h the creatinine o u t p u t . Tables 10.1 and 10.2 show modified nucleoside content i n urine and serum from healthy adults (refs 10,48). Work from our laboratory i n collaboration w i t h Drs. Brewer, Gehrke, and Waalkes (ref. 1) and others (refs. 3-6) have shown t h a t 1 eve1 s of urinary modified nucl eosi de excretion correlate w i t h stage of disease and response t o therapy. Elevated levels of urinary modified nucleosides i n cancer patients return t o normal levels soon after effective therapy. Elevated levels of modified urinary nucleosides and bases i n animals precede the appearance of tumor. Thomale and Nass (ref. 5) have studied the excretion of various breakdown products of t R N A by mice treated w i t h a carcinogen, 3-methylcholanthrene. The tumor developed i n s i t u i s palpable a f t e r sixteen weeks. Death usually occurs around the twenty-third week. Modified nucleosides of 24 hr urine samples were determined from the i n i t i a t i o n of the experiment u n t i l demise of the animals. By the seventh week, when the tumor was n o t diagnosable, excretion o f the nucleosides was elevated. In the sixteenth week, levels of the various nucleosides may be elevated a s much as 2- t o 4-fold above those of
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TABLE 10.1 M o d i f i e d Nucl e o s i des creatinine) N $ m1A
PCNR mlI m lG ac4C m2G mG; t6A B-A1 BA
49 42 39 49 22 32 47 46 20 36
Male
and
p-AIBA
Content
X
S.D.
N
23.3 1.70 1.08 1.36 0.88 0.52 0.39 1.27 0.58 6.14
2.70 0.32 0.24 0.21 0.16 0.08 0.09 0.17 0.10 3.59
54 38 37 47 15 23 41 46 11 120*
in
Urine
Femgl e
(nmol / w o l
X
S.D.
27.0 1.94 1.10 1.34 0.84 0.56 0.39 1.30 0.73 5.8
5.21 0.42 0.38 0.39 0.29 0.12 0.15 0.41 0.11 4.1
N , number o f subjects; X , mean; , standard d e v i a t i o n . These values are very s i m i l i a r t o those reported e a r l i e r ( r e f . 10 and 48) except t h a t t h e values for t 6 A have been corrected by a f a c t o r o f 0.55 due t o e a r l i e r discrepancy w i t h the standard. These corrected values are very s i m i l a r t o those determined by radioimmunoassay ( r e f . 53). We a r e g r a t e f u l t o Dr. Barbara Vold and Dr. Eckhard Schlimme f o g p r o v i d i n g authentic t 6 A . Males 18-59 years and females 18-60 years o l d . From Kuo e t a l . ( r e f . 51).
untreated control s. The p r e c i s e mol e c u l a r mechanism which g i v e s r i s e t o t h i s l a r g e e x c r e t i o n has n o t been a s c e r t a i n e d . We e x p l o r e d whether s i m i l a r e a r l y changes i n abnormal excret i o n o f m o d i f i e d n u c l e o s i d e s a r e e v i d e n t i n humans b e f o r e c l i n i c a l m a n i f e s t a t i o n s o f cancer become apparent. T h i s may be u s e f u l f o r d e t e c t i n g p r e c l in i c a l b i ochemi c a l changes p r e d i c t i v e o f f u t u r e neoplastic manifestations. We d e s c r i b e here r e s u l t s o f o u r ongoing s t u d i e s on asbestos-exposed workers who a r e a t h i g h n e o p l a s t i c r i s k and s u b j e c t s i n f e c t e d w i t h H I V , A I D S v i r u s . The a b b r e v i a t i o n s used a r e : Pseudouridine ($), l - m e t h y l adenosine (m1A) ; 2 - p y r i done-5-carboxami de-Nl-ri b o f u r a n o s i de (PCNR, a d e g r a d a t i o n p r o d u c t o f N A D t ) ; l-methyl i n o s i n e (mlI); l-methylguanosi ne (m1G) ; N2-methyl guanosi ne (mZG) ; N2N2-dimethylguanosine (m;G) ; N4-acetyl c y t i d i ne (ac4C) ; N6-threonyl adenosi ne ( t 6 A ) and p a m i n o i s o b u t y r i c a c i d (p-AIBA)
'
.
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10.2 RESULTS AND DISCUSSION 10.2.1 Asbestos EXDOSUre and M o d i f i e d Nucleosides Asbestos-associ a t e d diseases a r e o f g r e a t concern i n *nany countries. The c u r r e n t i n c r e a s e i n t h e i n c i d e n c e o f t h e s e diseases n o t e d i n t h e U n i t e d S t a t e s appears t o be r e l a t e d t o t h e l a r g e number of i n d i v i d u a l s who o v e r t h e p a s t t h r e e o r f o u r decades have been employed i n work environments i n which t h e r e was p o t e n t i a l f o r exposure t o hazardous l e v e l s o f a i r b o r n e asbestos fibers. I t has been e s t i m a t e d t h a t , s i n c e t h e 1940's, more t h a n 25 m i 11 i o n persons have experienced exposure t o asbestos a t t h e i r A l o n g p e r i o d o f l a t e n c y between t h e places o f work ( r e f . 15). onset of exposure and t h e c l i n i c a l m a n i f e s t a t i o n o f d i s e a s e i s one o f t h e c h a r a c t e r i s t i c s o f asbestos-associ a t e d d i s o r d e r s ( r e f . 16). T h i s a p p l i e s p a r t i c u l a r l y t o t h e m a l i g n a n t diseases, such as l u n g cancer, p l e u r a l and p e r i t o n e a l mesothel ioma, which have been s t r o n g l y a s s o c i a t e d w i t h asbestos i n e p i d e m i o l o g i c a l s t u d i e s ( r e f . 17). I n general, t h e d i a g n o s i s o f a s b e s t o s - r e l a t e d cancer i s made a t a stage when t h e disease i s f a r advanced, and t h e t h e r a p e u t i c p o s s i b i l i t i e s are very l i m i t e d . Because o f t h i s s i t u a t i o n and because o f t h e p u b l i c h e a l t h problem t h a t asbestos-i nduced diseases pose i n s o c i e t y , t h e r e i s an u r g e n t need t o develop new methods t o i d e n t i f y i n d i v i d u a l s who may be a t h i g h r i s k o f developing t h e s e types o f o c c u p a t i o n a l cancers, and t o i n c r e a s e t h e p o t e n t i a l f o r e a r l y d i a g n o s i s and more e f f e c t i v e t r e a t m e n t . The term "asbestos" i s used t o d e s c r i b e a group o f n a t u r a l l y o c c u r r i n g f i b r o u s s i 1 ic a t e s and a r e c h a r a c t e r i z e d by g r e a t t e n s i 1e s t r e n g t h and r e s i s t a n c e t o chemicals and heat. The d e f i n i t i o n o f asbestos i s l i m i t e d here t o t h e f i b r o u s m i n e r a l s o f t h e s e r p e n t i n e and amphibole s e r i e s A s b e s t i f o r m m i n e r a l s can be c a t e g o r i z e d i n t o two m a j o r subd i v i s i o n s , namely c h r y s o t i l e , which belongs t o t h e s e r p e n t i n e s e r i e s and t h e amphiboles, which i n c l u d e c r o c i d o l i t e , a c t i n o l i t e t r e m o l i t e , amosite and a n t h o p h y l l i t e . The c r y s t a l l i n e s t r u c t u r e s of b o t h c h r y s o t i l e and amphiboles have been c l a r i f i e d . C h r y s o t i l e c o n s i s t s o f a l a y e r of magnesium oxide-hydroxide octahedra bonded t o a layer o f s i l i c o n dioxide tetrahedra. The s h e e t l i k e l a y e r tends t o r o l l i t s e l f i n t o a h o l l o w t u b e w i t h t h e magnesium hydrox-
.
TABLE 10.2 Modified Nucleosides and B-AIBA Content in Serum pmol/ml N $
m1I ml G ac4C m2G m:G t6A 8-AIBA
37 21 18 29 27 19 10 20
X
2810 41.5 41.0 71.6 19.8 47.1 38.2 2140
Ma1 e nmollpmol of creati nine -
U
523 9.80 17.1 18.9 7.59 20.0 12.6 960
X
40.3 0.61 0.61 1.04 0.29 0.73 0.59 32.4
pmol /ml U
8.07 0.16 0.27 0.27 0.12 0.40 0.18 14.7
N
48 35 16 36 33 29 19 27
T
2520 42.8 56.9 61.6 18.9 46.9 32.5 1680
Femal e nmollpmol o f creatinine U
538 17.2 18.4 20.1 5.72 21.4 10.5 684
-
X
46.3 0.76 1.15 1.11 0.35 0.91 0.62 30.7
U
12.5 0.29 0.55 0.35 0.13 0.50 0.23 12.5
D e t a i l s a r e same as f o r Table 9 . 1 . Males 19-56 years and females 21-52 years o l d . C r e a t i n i n e i n serum (males 13.8 nmol/ml) was determined by HPLC ( r e f . 52). 70.8 2 11.1; females 56.5
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ide on the outer surface. In contrast, amphiboles consist of doubl e chains of 1 inked si 1 i con-oxygen tetrahedra posi t i oned para1 1el t o the vertical crystal 1 ographi c axis and bound 1a t e r a l l y by m e t a l l i c ions. The chemical composition of the two asbestos types a l s o d i f f e r i n t h a t chrysotile contains l e s s s i l i c a and iron oxides than the amphiboles, b u t has a higher content of magnesium than the amphiboles ( r e f . 18). Asbestos-contai n i ng materi a1 s have been used extensively i n industry since i t s commercial introduction i n the l a t e 19th Century. I t i s estimated t h a t some 3,000 types of products contain some form of asbestos. One of i t s most important uses has been as a component i n heat and f r o s t insulation, and asbestos i s therefore often encountered i n s h i p b u i l d i n g and r e p a i r , construct i o n , power production, chemical manufacturing and i n the manufact u r i n g of automobile brakes ( r e f . 19). Adverse health e f f e c t s of inhaled asbestos f i b e r s began t o appear among occupational ly-exposed groups a t the e a r l y p a r t of the century. I t was subsequently shown t h a t inhaled asbestos f i b e r s can be retained i n the l u n g t i s s u e , where they can be i d e n t i f i e d e i t h e r by l i g h t microscopy, phase contrast microscopy, and electron microscopy, depending upon f i b e r s i z e and s t r u c t u r e ( r e f s . 20-22) . Asbestosis, i . e . , i n t e r s t i t i a l pulmonary f i b r o s i s , was the f i r s t disease related t o exposure t o airborne a s b e s t o s . ' I t i s s t i l l the most common manifestation of asbestos-induced e f f e c t s among occupational ly-exposed individuals. There i s usually a 1 atency period of approximately ten years before radiographic abnormalities occur. These are characterized radiographically by l i n e a r and r e t i c u l a r opacities affecting the middle and lower l u n g f i e l d s . I t i s also known t h a t a group of conditions of the pleura such a s thickening and c a l c i f i c a t i o n can be induced by asbestos ( r e f . 23).\ The f i r s t indications t h a t asbestos exposure m i g h t r e s u l t i n cancer appeared i n the 1930's, when a few cases of, 1 ung cancer were reported i n persons w i t h asbestosis ( r e f . 2 4 ) . I t was subsequently shown, i n epidemiological s t u d i e s , t h a t populations of individuals w i t h a h i s t o r y of occupational exposure t o asbestos
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have h i g h m o r t a l i t y r a t e s o f l u n g cancer ( r e f . 25-27). I t was a l s o c l a r i f i e d t h a t asbestos seems t o e x e r t i t s e f f e c t s y n e r g i s t i c a l l y w i t h tobacco smoke, and t h a t asbestos i n s u l a t i o n workers who smoke c i g a r e t t e s r u n t h e r i s k o f an e x t r a o r d i n a r i l y h i g h m o r t a l i t y r a t e i n l u n g cancer ( r e f . 28, 29). However, t h e r e i s some e v i dence, based. upon a small number o f d a t a p o i n t s , t h a t asbestos may i n c r e a s e t h e r i s k o f l u n g cancer even i n nonsmoking i n d i v i d u a l s . I t was r e c e n t l y observed t h a t t h e m o r t a l i t y o f l u n g cancer decreased s i g n i f i c a n t l y among asbestos workers who d i s c o n t i n u e c i g a r e t t e smoking as compared t o t h o s e who c o n t i n u e t h i s h a b i t ( r e f . 30). Although l u n g cancer may be t h e most common neoplasm among asbestos workers, m a l i g n a n t mesothel ioma o f t h e p l e u r a and p e r i toneum i s t h e most a s b e s t o s - s p e c i f i c m a l i g n a n t d i s e a s e ( r e f . 31). An e x t r e m e l y r a r e tumor i n t h e general p o p u l a t i o n , i t accounts f o r a p p r o x i m a t e l y 8% o f a l l deaths o f asbestos i n s u l a t i o n workers, a c c o r d i n g t o some s t u d i e s ( r e f . 17). Survival time i s usually l e s s t h a n a y e a r a f t e r d i a g n o s i s w i t h c u r r e n t l y a v a i l a b l e diagnost i c methods and t r e a t m e n t m o d a l i t i e s . I t i s o f i n t e r e s t t o note t h a t c i g a r e t t e smoking does n o t appear t o be r e l a t e d t o t h e r i s k o f devel o p i ng mesothel ioma ( r e f . 29) A c l e a r dose response r e l a t i o n s h i p , which seems t o e x i s t f o r asbestosis, has n o t been i d e n t i f i e d w i t h t h e same degree o f c e r t a i n t y f o r e i t h e r l u n g cancer o r mesothel ioma. I n a d d i t i o n t o l u n g cancer and mesothelioma, o t h e r neoplasms have a1 so been a s s o c i a t e d w i t h o c c u p a t i o n a l exposure t o asbestos. These i n c l u d e c o l on-rectum cancer, esophageal and stomach cancer, as w e l l as cancer o f t h e oropharynx and l a r y n x ( r e f s . 17, 32, 33). I t should be emphasized, however, t h a t t h e degree o f evidence f o r t h e a s s o c i a t i o n between t h e l a t t e r groups o f cancers and asbestosexposure i s l e s s s t r o n g than f o r l u n g cancer and mesothelioma ( r e f . 34). As m e n t i o n e d , a s b e s t o s - r e l a t e d diseases, including the asbestos-associated cancers, usual l y become c l i n i c a l l y mani f e s t o n l y a f t e r a l o n g t i m e l a p s e f r o m onset o f exposure. For asbestosis, t h i s i s u s u a l l y a decade o r so, b u t f o r t h e asbestosr e l a t e d n e o p l a s t i c diseases, two t o f o u r decades i s u s u a l l y t h e r u l e , D u r i n g t h a t l a t e n c y p e r i o d , t h e i n d i v i d u a l may e i t h e r have
.
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radiographic evidence of asbestosis or may be entirely without symptoms or signs of exposure. I n either case, the exposed individual does not manifest any sign or symptom that would make possible the identification of the individual's high neoplastic risk. The protracted period of latency poses a difficult problem for early detection and, especially, for successful therapy. The information obtained in retrospective prospective epidemiological investigations of asbestos insulation workers provides evidence for the serious consequences that may occur when there has been a delay in establishing a cause-effect relationship, and when the biological and biochemical events during the 1 atency period remain poorly understood. Because of this situation, there is an urgent need for developing new diagnostic methods which could assist i n the identification of pathophysiological changes at a stage when early intervention might be of added advantage. We report observations of the urinary excretion patterns of modified nucleosides in patients with malignant mesothel ioma as a possible approach for the early detection of this disease. The first pilot study addressed the question whether patients w.ith asbestos-associated ma1 ignant mesothel ioma excrete elevated levels of modified nucleosides i n their urine. Eight patients with mesothelioma were studied and the results showed that several nucleosides were elevated, with $ elevated in all patients, Table 10.3 (ref. 35). Subsequently, similar results were obtained from additional seven patients with ma1 ignant mesothel ioma (Unpublished observation). These results indicate that mesothelioma produces elevated excretion of nucleosides and that this may be an additional diagnostic tool . No fa1 se-negative resul ts were observed i n this small group of patients with mesothelioma and the laboratory investigators were able to identify all as "cancers" on the basis of the quantitation of nucleosides. A recent stu y has shown increased excretion of $ and hypoxanthine in nude mice transplanted with mesothelioma (ref. 49). Excretion of $ s related to the growth of mesothelioma and an increased excretion commences at a time when the tumor is just measurable. The potentially useful predictive value of study ng nucleo-
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side excretion patterns i s reflected i n the i n i t i a l p i l o t study. Ten of 13 workers whose onset of asbestos exposure preceded the examination date by 30 t o 40 years had elevated levels of nucleosides. Elevated excretion of pb was the p r i n c i p a l a b n o r ma l i t y . This i s of interest since this gr oup of workers constitutes a population a t p a r t i c u l a r l y h i g h risk of neoplastic disease development (ref. 17). Follow-up information t o date on 3 of the 10 workers gave evidence of cancer. One developed lperi toneal mesothe1 i oma. A second study was therefore undertaken t o investigate whether apparently well asbestos insulation workers w i t h l o n g histories of asbestos exposure (30 years or longer), b u t w i t h o u t current evidence of cancer, would show unusual urinary excretion patterns of modified nucleosides. A group of 47 male individuals TABLE 10.3
Elevated Modified Nucleosides i n Patients w i t h Mesothelioma Patient 1 2 3 4 5 6 7 8 Normal
Sex
Marker Levels (nmol/fimol creatini ne) $
M M M M M
49.2 41.1 114.5 38.6 32.9 M 38.4 M 48.3 M 36.6 M 22.4
mlA
1.80 1.84 4.85 2.97 NRa 3.23 2.78 2.22 1.77
PCNR
2.81 1.74 3.75 1.90 1.97 1.90 1.02 1.56 0.87
m'I
mlG
mzG
m:G
B-AIBA
2.30 2.85 5.50 2.16 1.69 1.94 1.63 1.31 1.15
0.87 1.20 3.76 NR 1.44 2.15 1.30 0.79 1.06
0.54 0.44 1.46 0.53 0.73 0.73 0.40 0.68 0.35
2.19 2.26 4.32 1.96 1.72 2.24 1.44 1.30 1.2
6.0 4.0 21.0 30.0 3.0 4.0 9.4 4.0 4.27
(k2.10)b(*0.29) (i0.25) (i0.27) F
26.7
1.76
1.05
1.18
(e4.5) (k0.48) (+0.26)(e0.39) aNR, N o t R e s o l v e d hean S.D.
(k0.07) (20.15) (k1.93)
1.07 0.41
1.44
5.8
(k0. 12) (k0.38) (k4.11)
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was examined. They had been selected from a population of close to 2,000 workers from whom we have collected urine samples in the course of cl i ni cal examinations. The results of this feasi bi 1 i ty study showed that asbestos insulation workers exhibit significantly higher levels of seven of the investigated nucleosides when compared to .b control group. Twenty-seven (57%) of the asbestos workers had three or more nucleosides elevated, indicating a highly abnormal excretion profile (ref. 36). ' An increasing severity of radiographic a1 terati ons was associated with a greater frequency of elevated nucleosides, especially with mrA, mlI, mlG, and m$G. Duration since onset o f exposure was directly related to $, mlI, and m$G. We have also examined d
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Because of the possi bi 1i t y of re1 a t i ng .the nucl e o s i d e l e v e l s t o e x t e n s i v e c l i n i c a l and l a b o r a t o r y information a v a i l a b l e on this high r i s k population, i t i s a n t i c i p a t e d t h a t important information w i l l be obtained about determinants of nucleoside l e v e l s and m u l t i p l e f a c t o r i n t e r a c t i o n s which may g i v e c l u e s t o p r e v e n t i v e measures ( r e f . 38). 10.2.2 Urinary Excretion of Modified Nucleosides i n P a t i e n t s w i t h Various Manifestations o f I n f e c t i o n with HIV The d i s e a s e , Acquired Immune Deficiency Syndrome (AIDS) has r e c e n t l y approached epidemic proportions i n many p a r t s of the world. AIDS i s c h a r a c t e r i z e d by severe immune depression and s u s c e p t i b i l i t y t o neoplasms and o p p o r t u n i s t i c i n f e c t i o n s . Recent evidence s t r o n g l y i m p l i c a t e s a lymphotropic r e t r o v i r u s , HIV, a s the primary e t i o l o g i c agent of AIDS ( r e f . 39-42). During a f i v e y e a r period, 5-19% of i n d i v i d u a l s with HIV a n t i b o d i e s developed AIDS and there was a l a t e n c y period o f 1-5 y e a r s between i n f e c t i o n and development of d i s e a s e . Pathognomonic immunological o r biochemical a b e r r a t i o n s t h a t d e l i n e a t e t h i s syndrome a r e not known. We have observed e a r l ier e l evated 1eve1 s o f modi f i ed nucleosides and j-AIBA i n u r i n e from p a t i e n t s w i t h AIDS and i n a male homosexual population from New York C i t y ( r e f . 43). A goal of o u r s t u d y i s t o explore whether i t i s p o s s i b l e t o i d e n t i f y immunological o r biochemical abnormal i t i e s i n persons with HIV i n f e c t i o n s t h a t may i d e n t i f y s u b j e c t s with p r e d i s p o s i t i o n f o r development of AIDS. Conceivably, such a s t u d y might allow selection of subjects f o r early therapeutic intervention. The s u b j e c t s (20-52 y e a r s o l d ) f a l l i n t o f o u r c a t e g o r i e s : 1) T h i r t y - f i v e h e a l t h y homosexual men (HHS), none of whom a r e c h r o n i c c a r r i e r s of h e p a t i t i s B s u r f a c e a n t i g e n ; a n a l y s i s of s t o r e d frozen serum by an enzyme-linked immunosorbent assay (ELISA) f o r HIV antibody divided t h i s group r e t r o s p e c t i v e l y i n t o two subcategori e s : 20 h e a l t h y homosexual men who were s e r o n e g a t i v e (HHS-) f o r antibody t o the HIV (ELISA r a t i o l e s s than 3.00) a t the time of the immunol ogi c'al and c l i n i cal eval u a t i ons and nucl e o s i de d e t e r minations and 15 heal t h y homosexual men who were , s e r o p o s i t i v e (HHS+) f o r antibody t o the HIV (ELISA r a t i o g r e a t e r than o r equal t o 5.00) a t the time of e v a l u a t i o n s ; 2) 38 asymptomatic homosexual
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men w i t h Chronic lymphadenopathy syndrome (CLS), defined a s d i s c r e t e lymph nodes i n two o r more non-inguinal s i t e s of three o r more months duration ( r e f . 44); none of t h e s e p a t i e n t s had symptoms o r f i n d i n g s of fevers, n i g h t sweats, w e i g h t l o s s , f a t i g u e , d i a r r h e a , Herpes z o s t e r , Herpes simplex o r i d i o p a t h i c thrombocytopenia; 3) 14 s u b j e c t s w i t h AIDS r e l a t e d complex (ARC) defined a s a syndrome of i n t e r m i t t e n t o r continuous fevers >38.50 f o r > one month, watery d i a r r h e a of two weeks d u r a t i o n f o r which no o t h e r e t i o l o g i e s were e s t a b l i s h e d , unexplained weight l o s s of >15 pounds o r >lo% of the body weight o r minor o p p o r t u n i s t i c infect i o n s such a s oral c a n d i d i a s i s o r Herpes z o s t e r ; and 4) 21 homosexual men w i t h AIDS who met epidemiologic s u r v e i l l a n c e d e f i n i t i o n o f the Centers f o r Disease Control. Only one of the ARC p a t i e n t s had noted s i g n i f i c a n t weight l o s s d u r i n g the weeks p r i o r t o the study. Of the p a t i e n t s w i t h AIDS, twelve had o p p o r t u n i s t i c i n f e c t i o n s (01), and nine had Kaposi's sarcoma (KS). The control population consisted of 52 healthy a d u l t heterosexual males ranging i n age from 18-60 y e a r s o l d . Assays f o r a n t i b o d i e s t o HIV were performed on s t o r e d frozen serum samples using ELISA (ref. 4 5 ) . P a t i e n t s w i t h AIDS, CLS, ARC and asymptomatic homosexuals (HHS) excreted increased amounts of markers i n the urine ( r e f . 4 6 ) . The numbers of abnormally excreted markers i n the urine of p a t i e n t s increased by d i s e a s e category ( F i g . 10.1). P a t i e n t s w i t h AIDS had g r e a t e r e x c r e t i o n of nucleosides t h a n any o t h e r group; 74% o f the p a t i e n t s w i t h CLS had a t l e a s t one abnormal marker, and 42% had t h r e e o r more e l e v a t i o n s ( F i g . 10.2). In c o n t r a s t , over 90% of p a t i e n t s w i t h AIDS o r ARC had one abnormality i n nucleoside excretion and over 75% of persons w i t h AIDS o r ARC had abnormal excretion of t h r e e nucleosides. Interestingly, 80% of the HHS had elevated e x c r e t i o n of one o r more markers (Fig. 10.3). HHS t h a t were p o s i t i v e f o r HIV antibody (HHS(+)), e x h i b i t e d a s i g n i f i c a n t l y different nucleoside excretion p a t t e r n than those who were negative f o r HIV antibody Only 30% of the HHS(-) i n d i v i d u a l s excre\ted abnormal (HHS(-)). amounts of the three markers while 60% of HHS (+) s u b j e c t s exc r e t e d abnormal amounts of t h r e e nucleosides. Amongst a l l markers, t 6 A was c o n s i s t e n t l y elevated i n over 70% of the HHS(+)
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F i g u r e 10.1. Abnormal e x c r e t i o n o f m o d i f i e d n u c l e o s i d e s by pat i e n t s w i t h A I D S w i t h K a p o s i ' s sarcoma KS), A I D S w i t h o p p o r t u n i s t i c i n f e c t i o n s (01), persons w i t h t h e A DS-related com l e x (ARC o r t h e g e n e r a l i z e d c h r o n i c lymphadenopathy syndrome ([LS and I V a n t i b o d y - p o s i t i v e asymptomatic homosexual men (HHS+). T e abnormal values r e p r e s e n t c o n c e n t r a t i o n s more t h a n two s t a n d a r d d e v i a t i o n s g r e a t e r t h a n t h e mean f o r t h e heterosexual c o n t r o l s u b j e c t s . Modi f i e d n u c l eosides were determined by HPLC u s i n g a Radi a1 -Pak c a r t r i d g e ( r e f . 10). t3-AIBA ( b o t h f r e e and bound) was analyzed by HPLC u s i n g f l u o r e s c e n c e d e t e c t i o n o f t h e e l u a t e w i t h o - p h t h a l a l dehyde ( r e f . 48). M o d i f i e d n u c l e o s i des and B-AIBA were expressed relative t o creatinine.
I
1,
A
The g r e a t e s t d i f f e r e n c e s and i n 58% o f t h e HHS(-) s u b j e c t s . between s e r o n e g a t i v e and s e r o p o s i t i v e HHS were seen w i t h m'l, ac4C, #, and m$G. There were a l s o d i f f e r e n c e s i n Qe e x t e n t o f e l e v a t i o n o f v a r i o u s markers determined by Z values, between
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0.0
2.0
4.0
6.0
8.0
10.0
NUMBER OF ABNORMALITIES
Figure 10.2. Numbers of abnormalities in excretion of nucleosides amongst members o f various clinical groups. CLS, KS, and 01. greater excretion of mlI, ac4C and m2G, t h a n HHS(-) subjects (Column 1). However, with the exception of m l G , no such positive correlation was evident between HHS(+) subjects and CLS (Column 2 ) . The most striking differences were between the CLS and ARC groups in which ARC-patients excreted greater amounts of seven of the ten urinary markers (Column 3 ) . Whereas there were few differences between ARC subjects and those with AIDS with Kaposi's sarcoma or AIDS with opportunistic infections (Columns 4 a n d 5 ) . In an earlier sbudy, two of the modified nucleosides, $ and mzG, correlated significantly with the number of s i t e s of palpable lymph nodes. The correlation remained significant w h & b o t h
loo
J
z U
75
-
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HETEROSEXUAL &---A HHS (-1 HHS (+I
0
z U
50
I-
z W
0
5 n
25
0
0.0
2.0
4.0
6.0
8.0
10.0
NUMBER OF ABNORMALITIES
F i g u r e 10.3. Comparison o f m o d i f i e d n u c l e o s j d e e x c r e t i o n b y H I V a n t i body-negative (HHS-) and H I V a n t i body-posi t i v e (HHS+ asymptom a t i c homosexual men. Both groups d i f f e r from heal t h y . e t e r o s e x ual c o n t r o l s and t h e HHS+ s u b j e c t s have more a b n o r m - a l i t i e s than HHS- s u b j e c t s .
b
i n g u i n a l and submandi b u l a r lymph nodes were excluded [ r e f . 43). M o d i f i e d nucleosides, $, mlI, m l G , ac4C, m*G, m$G, t 6 A and PAIBA, were a l s o determined i n t h e matched s e r a of s u b j e c t s . In general i n serum, e l e v a t e d e x c r e t i o n o f t h e s e markers s i m i l a r t o t h a t i n u r i n e was observed i n HHS s u b j e c t s and t h o s e w i t h CLS, ARC, o r A I D S . However, when t h e mean values o f markers i n serum i n v a r i o u s d i a g n o s t i c groups were compared, no s i g n i f i c a n t c o r r e l a t i o n between serum markers and HHS s u b j e c t s and s u b j e c t s w i t h CLS and t h o s e w i t h ARC was e v i d e n t . Therefore, e x c r e t i o n o f markers i n u r i n e appear t o be more r e l e v a n t t h a n serum f o r t h e s e studies. The e f f e c t o f c h r o n i c v i r a l i n f e c t i o n s and use o f i n t r a v e n o u s drugs on t h e e x c r e t i o n o f v a r i o u s m o d i f i e d n u c l e o s i d e s has n o t been s t u d i e d i n d e t a i l . Chick e m b r y o f i b r o b l a s t s i n f e c t e d w i t h
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TABLE 10.4 C o r r e l a t i o n Between Markers and D i a g n o s t i c Groups (Ref. 43) \
H I V (-) HIV,(+) $
mlA PCNR m1 I m1 G ac4C mzG m$G t6A j-AIBA
NSa NS NS p=O. 005 NS p=0.02 p=O. 04 NS NS NS
VS.
D i a a n o s t i c Grouos HHS(+) VS. CLS V S . c LS ARC NS NS NS NS p=O. 024 NS NS NS NS NS
p=o. 02 NS p=o. 001 p=o. 002 NS p=O. 007 p=o. 02 p=O.Ol p=o. 0 1
NS
ARC V S . KS
ARC 01
NS NS NS NS NS NS
NS NS NS p=O. 04 NS NS NS NS NS NS
NS NS NS NS
VS.
aNS, No significant correlation ( p > 0.05).
Rous sarcoma v i r u s e x c r e t e e l e v a t e d l e v e l s o f $ ( r e f . 47). We have observed t h a t i n f o u r a d u l t male s u b j e c t s p o s i t i v e f o r h e p a t i t i s B antigen, o n l y one s u b j e c t e x c r e t e d e l e v a t e d l e v e l s o f markers. I n d i v i d u a l s a t h i g h r i s k f o r AIDS, u s e r s o f m a r i j u a n a and cocaine had s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s between a c u m u l a t i v e i n d e x o f o v e r - a l l drug use and e x c r e t i o n o f PCNR and m2G. Users o f amyl n i t r i t e and e t h y l c h l o r i d e e x c r e t e d l o w e r l e v e l s o f markers ( r e f . 43). I n a recent study i n c o l l a b o r a t i o n w i t h D r . Joyce Wallace ( r e f . 50), we have found t h a t asymptoma i c women i n a methadone maintenance program from New York C i Y I p o s i t i v e f o r H I V antibody, e x c r e t e d e l e v a t e d l e v e l s o f m o d i f ed nucleosides compared t o women who were n e g a t i v e f o r a n t i b o d y t o HIV. The mechanisms t h a t produce abnormal n u c l e o s i d e e x c r e t i o n i n asymptomatic a d u l t s and i n s u b j e c t s w i t h H I V d i s e a s e a r e unknown. A p r o s p e c t i v e s t u d y i s under progress t o a s c e r t a i n t h e u s e f u l n e s s o f measuring m o d i f i e d n u c l e o s i d e e x c r e t i o n t o i d e n t i f y s u b j e c t s who w i l l progress t o ARC o r A I D S and t o m o n i t o r response t o treatment.
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10.3
1.
2.
3.
4.
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1
I )
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A. F i s c h b e i n , 0. K. Sharma, I.J. S e l i k o f f and E. Borek, Urinary excretion o f modified nucleosides i n p a t i e n t s w i t h m a l i g n a n t mesothelioma, Cancer Res., 43 (1983) 2971-2974. A. F i s c h b e i n , 0. K. Sharma, S. J. Solomon, F. Buschman, G. A p e l l , M. Kohn, I.J. S e l i k o f f , J. G. Bekesi and E. Borek, T r a n s f e r RNA breakdown p r o d u c t s i n t h e u r i n e o f asbestos workers Cancer D e t e c t . Prev., 7 (1984) 247-252. S. J. Solomon, A. Fischbein, 0. K. Sharma and E. Borek, Modifie'd n u c l e o s i d e s i n asbestos workers a t h i g h r i s k o f m a l i g n a n t disease: R e s u l t s of a r e l i m i n a r y s t u d appl i n d i s c r i m i n a n t a n a l y s i s , B r . J. Pndust. Med., 43 (18857 560-562. A. Fischbein, 0. K. Sharma and E. Borek, Modi f ied n u c l e o s i d e s and e a r l y d e t e c t i o n o f o c c u p a t i o n a l cancer: A c h a l l e n g e f o r t h e f u t u r e , M t . S i n a i J. Med., 52 (1985) 480-483. F. Barre-Sinoussi, J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. A x l e r - B l i n , F. VezinetBrun, C. R o l l z i o u x , W. Rozenbaum and L. Montagnier, I s o l a t i o n o f a T-1 mphotro i c r e t r o v i r u s f r o m a a t i e n t a t r i s k f o r Acquired mmune g e f i c i e n c y Syndrome (APDS) , Science, 220 (1983) 868-871. R. C. G a l l o , S. Z. Salahuddin M. Popovic, G. M. Shearer, M. Kaplan, B.F. Haynes, T. J . P a i k e r , R. R e d f i e l d , J. Oleske, B. S a f a i , G. White, P. F o s t e r and P. D. Markham, Frequent d e t e c t i o n and i s o l a t i o n o f c y t o p a t h i c r e t r o v i r u s e s (HTLV111) f r o m p a t i e n t s w i t h A I D S and a t r i s k f o r A I D S , Science, 224 (1984) 500-503. S. H. Landesman, H. M. Ginzbur and S. H. Weiss, The A I D S epidemic, New Engl J. Med., 318 (1985) 521-525 P. S. S a r i n and R. C. Gallo, Human T-lymphotropic r e t r o v i r u s e s i n a d u l t T - c e l l leukemia-lym homa and ac u i r e d q1984) immune d e f i c i e n c y syndrome, J. C1 i n . fmmunol , 4 415-423. A. Fischbein, 0. K. Sharma, J. A. Valciukas, J. G. Bekesi, F. Buschman, G. A p e l l , M. A. Kohn, R. R. Boesch, A. T e i r s t e i n , I.J. S e l i k o f f and E. Borek, U r i n a r y e x c r e t i o n o f m o d i f i e d n u c l e o s i d e s i n p a t i e n t s w i t h a c q u i r e d immune d e f i c i e n c y syndrome and i n d i v i d u a l s a t h i h r i s k o f d e v e l o p i n g t h e s e (1985) 271-277. diseases, Cancer D e t e c t . Prev., Centers f o r Disease C o n t r o l : Persistent, eneral i z e d 1 mphadenopathy among homosexual males. MMWR 3 1 71982) 2492g1 S. H. Weiss, J. J . Goedert, M. G. Sarngadharan, A. J. Bodner, R . C. G a l l o and W . A. B l a t t n e r , Screening t e s t f o r HTLV-I11 ( A I D S agent) a n t i b o d i e s . [Specificity, sensitivity and appl ic a t i ons, JAMA, 253 (1985 221-225. E. Borek, 0. K. Sharma, F. Buschman, D. L. Cohn, K. A. Penley, F. N. Judson B. S. Dokozin, C. R. Horsburg, Jr., and C. H. K i r k p a t r i c k , A f t e r e d e x c r e t i o n o f m o d i f i e d n u c l e o s i d e s and O-Aminoisobutyric a c i d i n s u b j e c t s w i t h a c q u i r e d jmmune d e f i c i e n c y syndrome o r a t r i s k f o r ac u i r e d immune d e f i c i e n cy syndrome, Cancer Res., 46 (1986 2227-2561 F. Es o s i t o , T. Russo, R. Ammendo a, A. D u i l i o , F. S a l v a t o r e and Cimino, Pseudouridine e x c r e t j o n and t r a n s f e r .RNA p r i m e r s f o r r e v e r s e t r a n s c r i p t a s e i n tumors o f r e t r o v i r a l o r i g i n , Cancer Res. , 45 (1985) 6260-6263.
i
40.
41. 42. 43.
44. 45. 46.
47.
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!
1.
1
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48. F. L. Buschman, G. Ape11 and 0: K..Sharma, High performance liquid chromatographic determination o f R-aminoisobutyric acid i n picomole range, J. Chromat. Biomedical Applications, 347 (1986) 129-136. 49. L . Buhl, C. Dragshold, P. Svendsen, E. Hage a\nd M. R. Buhl, Urinary h poxanthi ne and seudouridi ne as indicators . of tumor deve opment in m e s o d e l ioma-transplanted nude mice, Cancer Res., 45 (1985) 1159-1163. 50. J. I. Wallace, E. Borek, F. L. Buschman, J. Mann, S. Solomon and 0. K. Sharma, Elevated urinary. excretion of modified nucleosides in HIV positive asymptomatic women i n a methadone maintenance rogram, Abstract, I11 International Conference on Acquired mmunodeficiency Syndrome, Washington, D. C. (in 19871. . F . Cole, C.W. Gehrke, T.P. Waalkes and E . Borek, 51. !Te?'Kuo, Dual. co1 umn cationrexchange chromatographic method for 8-am1 no1 sobutyri c acid and p a l an1 ne i n bi ol ogi cal samples. Clin. Chem. 24 1978) 1373-1380. 52. W. L. Chiou, F: ..Pu and T. Prueksaritanont, XI11 Micro higherformance 1 1 quid chromatographic assay of creati ni ne in iological fluids using fixed or variable wavelength U V detector, J. Chromatogr. 277 (1983) 436-438. 53. B. S. Vold, D. E. Keith, Jr., and M. Slavik, Urine Levels of N-[g-(j-D-Ri bofuranosyl) purin-6-ylcarbamo 1 -L-threonine, N6 -(A* -isopentenyl)adenosine, and 2'0-met .y guanosine as determined by radi oimmunoassay for normal subjects and cancer patients , Cancer Research 42 (1982) 5265-5269.
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CHAPTER 10 MODIFIED NUCLEOSIDES AS BIOCHEMICAL MARKERS OF ASBESTOS EXPOSURE AND AIDS* OPENDRA K. WARMAl, P h . D . and ALF FISCHBEINZ, M . D . ' L a b o r a t o r y o f M o l e c u l a r B i o l o g y , AMC C a n c e r R e s e a r c h C e n t e r , 1600 P i e r c e S t r e e t , Denver, Colorado
2 D i v i s i o n o f E n v i r o n m e n t a l and O c c u p a t i o n a l W e d i c i n e , Mount S i n a i S c h o o l o f M e d i c i n e o f t h e C i t y U n i v e r s i t y o f New Y o r k , One G u s t a v e L . Levy P l a c e , New Y o r k , New Y o r k
TABLE OF CONTENTS
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . C321 10.2 Results and Discussion . . . . . . . . . . . . . . . . . C324 10.2.1 Asbestos Exposure and Modified Nucleosides . . . . C324 10.2.2 Urinary Excretion of Modified Nucleosides in Patients With Various Manifestations of Infection With H I V . . . . . . . . . . . . . . . . . . C331 10.3 References . . . . . . . . . . . . . . . . . . . . . . C337
10.1 INTRODUCTION Cancer p a t i e n t s and tumor-beari ng animals excrete in t h e i r urine increased amounts of modified purines and pyrimidines ( r e f s . 1-6). These modified nucleosides, synthesized a t the macromolecular level ( r e f . 7 ) , are primarily constituents of t R N A and t o a l e s s e r extent of other RNAs. When RNA i s catabolized, most of these modified nucleosides cannot be r e u t i l i z e d ; consequently they a r e excreted. Pioneering s t u d i e s o f Borek e t a l . ( r e f . 8) have suggested t h a t excretion of elevated amounts of modified nucl eosides in tumor-beari ng animal s resul t s from increased t R N A turnover r a t h e r than from c e l l death. The molecular mechanisms of elevated excretion a r e unclear. Extracts of neopl a s t i c t i s s u e s have aberrant t R N A methyltransferases ( r e f . 7 ) and i t has been suggested t h a t the high turnover of t R N A i s due t o rapid degradation of aberrantly modified tRNAs ( r e f s . 7 , 8 ) . * T h i s work was supported by USPHS Grants HL-32432, HD-20612 OH-02122, NIEHS Center Grant E S 00928, CDC Grant OH-02122 and a g i f t t o t h e AMC Cancer Research Center from G e r a l d M . Q u i a t .
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More recent studies indicate t h a t catabolism of other RNA species and altered RNA metabolism of host tissue (refs. 5, 9 ) may also contribute t o elevated urinary levels of modified nwleosides. The urinary excretion of modified nucleosides and j-AIBA (degradation product of thymine) expressed re1 a t i ve t o creati n i ne i s remarkably constant for normal 18-60 year o l d subjects (refs. 1, 10). Infants and children excrete lower levels o f modified nucleosides compared t o adults (ref. 2 ) . Patients suffering from non-cancerous diseases do n o t excrete significantly elevated levels of modified nucleosides (ref. 11), however, elevated excretion i s observed i n some patients w i t h acute hepatitis (ref. l l ) , g o u t and psoriasis (ref. 12). Children w i t h acute infections do n o t excrete significantly elevated levels of modified nucleosides when compared t o healthy children (ref. 13). S l i g h t elevations i n u r i n a r y modified nucleoside excretion from subjects w i t h bacterial pneumonia and significant elevations from some subjects w i t h urinary tract infections have been noticed ( r e f . 143. Therefore, urinary excretion of modified nucleosides should be interpreted w i t h caution i n subjects w i t h syndromes t h a t interfere w i t h the creatinine o u t p u t . Tables 10.1 and 10.2 show modified nucleoside content i n urine and serum from healthy adults (refs 10,48). Work from our laboratory i n collaboration w i t h Drs. Brewer, Gehrke, and Waalkes (ref. 1) and others (refs. 3-6) have shown t h a t 1 eve1 s of urinary modified nucl eosi de excretion correlate w i t h stage of disease and response t o therapy. Elevated levels of urinary modified nucleosides i n cancer patients return t o normal levels soon after effective therapy. Elevated levels of modified urinary nucleosides and bases i n animals precede the appearance of tumor. Thomale and Nass (ref. 5) have studied the excretion of various breakdown products of t R N A by mice treated w i t h a carcinogen, 3-methylcholanthrene. The tumor developed i n s i t u i s palpable a f t e r sixteen weeks. Death usually occurs around the twenty-third week. Modified nucleosides of 24 hr urine samples were determined from the i n i t i a t i o n of the experiment u n t i l demise of the animals. By the seventh week, when the tumor was n o t diagnosable, excretion o f the nucleosides was elevated. In the sixteenth week, levels of the various nucleosides may be elevated a s much as 2- t o 4-fold above those of
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TABLE 10.1 M o d i f i e d Nucl e o s i des creatinine) N $ m1A
PCNR mlI m lG ac4C m2G mG; t6A B-A1 BA
49 42 39 49 22 32 47 46 20 36
Male
and
p-AIBA
Content
X
S.D.
N
23.3 1.70 1.08 1.36 0.88 0.52 0.39 1.27 0.58 6.14
2.70 0.32 0.24 0.21 0.16 0.08 0.09 0.17 0.10 3.59
54 38 37 47 15 23 41 46 11 120*
in
Urine
Femgl e
(nmol / w o l
X
S.D.
27.0 1.94 1.10 1.34 0.84 0.56 0.39 1.30 0.73 5.8
5.21 0.42 0.38 0.39 0.29 0.12 0.15 0.41 0.11 4.1
N , number o f subjects; X , mean; , standard d e v i a t i o n . These values are very s i m i l i a r t o those reported e a r l i e r ( r e f . 10 and 48) except t h a t t h e values for t 6 A have been corrected by a f a c t o r o f 0.55 due t o e a r l i e r discrepancy w i t h the standard. These corrected values are very s i m i l a r t o those determined by radioimmunoassay ( r e f . 53). We a r e g r a t e f u l t o Dr. Barbara Vold and Dr. Eckhard Schlimme f o g p r o v i d i n g authentic t 6 A . Males 18-59 years and females 18-60 years o l d . From Kuo e t a l . ( r e f . 51).
untreated control s. The p r e c i s e mol e c u l a r mechanism which g i v e s r i s e t o t h i s l a r g e e x c r e t i o n has n o t been a s c e r t a i n e d . We e x p l o r e d whether s i m i l a r e a r l y changes i n abnormal excret i o n o f m o d i f i e d n u c l e o s i d e s a r e e v i d e n t i n humans b e f o r e c l i n i c a l m a n i f e s t a t i o n s o f cancer become apparent. T h i s may be u s e f u l f o r d e t e c t i n g p r e c l in i c a l b i ochemi c a l changes p r e d i c t i v e o f f u t u r e neoplastic manifestations. We d e s c r i b e here r e s u l t s o f o u r ongoing s t u d i e s on asbestos-exposed workers who a r e a t h i g h n e o p l a s t i c r i s k and s u b j e c t s i n f e c t e d w i t h H I V , A I D S v i r u s . The a b b r e v i a t i o n s used a r e : Pseudouridine ($), l - m e t h y l adenosine (m1A) ; 2 - p y r i done-5-carboxami de-Nl-ri b o f u r a n o s i de (PCNR, a d e g r a d a t i o n p r o d u c t o f N A D t ) ; l-methyl i n o s i n e (mlI); l-methylguanosi ne (m1G) ; N2-methyl guanosi ne (mZG) ; N2N2-dimethylguanosine (m;G) ; N4-acetyl c y t i d i ne (ac4C) ; N6-threonyl adenosi ne ( t 6 A ) and p a m i n o i s o b u t y r i c a c i d (p-AIBA)
'
.
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10.2 RESULTS AND DISCUSSION 10.2.1 Asbestos EXDOSUre and M o d i f i e d Nucleosides Asbestos-associ a t e d diseases a r e o f g r e a t concern i n *nany countries. The c u r r e n t i n c r e a s e i n t h e i n c i d e n c e o f t h e s e diseases n o t e d i n t h e U n i t e d S t a t e s appears t o be r e l a t e d t o t h e l a r g e number of i n d i v i d u a l s who o v e r t h e p a s t t h r e e o r f o u r decades have been employed i n work environments i n which t h e r e was p o t e n t i a l f o r exposure t o hazardous l e v e l s o f a i r b o r n e asbestos fibers. I t has been e s t i m a t e d t h a t , s i n c e t h e 1940's, more t h a n 25 m i 11 i o n persons have experienced exposure t o asbestos a t t h e i r A l o n g p e r i o d o f l a t e n c y between t h e places o f work ( r e f . 15). onset of exposure and t h e c l i n i c a l m a n i f e s t a t i o n o f d i s e a s e i s one o f t h e c h a r a c t e r i s t i c s o f asbestos-associ a t e d d i s o r d e r s ( r e f . 16). T h i s a p p l i e s p a r t i c u l a r l y t o t h e m a l i g n a n t diseases, such as l u n g cancer, p l e u r a l and p e r i t o n e a l mesothel ioma, which have been s t r o n g l y a s s o c i a t e d w i t h asbestos i n e p i d e m i o l o g i c a l s t u d i e s ( r e f . 17). I n general, t h e d i a g n o s i s o f a s b e s t o s - r e l a t e d cancer i s made a t a stage when t h e disease i s f a r advanced, and t h e t h e r a p e u t i c p o s s i b i l i t i e s are very l i m i t e d . Because o f t h i s s i t u a t i o n and because o f t h e p u b l i c h e a l t h problem t h a t asbestos-i nduced diseases pose i n s o c i e t y , t h e r e i s an u r g e n t need t o develop new methods t o i d e n t i f y i n d i v i d u a l s who may be a t h i g h r i s k o f developing t h e s e types o f o c c u p a t i o n a l cancers, and t o i n c r e a s e t h e p o t e n t i a l f o r e a r l y d i a g n o s i s and more e f f e c t i v e t r e a t m e n t . The term "asbestos" i s used t o d e s c r i b e a group o f n a t u r a l l y o c c u r r i n g f i b r o u s s i 1 ic a t e s and a r e c h a r a c t e r i z e d by g r e a t t e n s i 1e s t r e n g t h and r e s i s t a n c e t o chemicals and heat. The d e f i n i t i o n o f asbestos i s l i m i t e d here t o t h e f i b r o u s m i n e r a l s o f t h e s e r p e n t i n e and amphibole s e r i e s A s b e s t i f o r m m i n e r a l s can be c a t e g o r i z e d i n t o two m a j o r subd i v i s i o n s , namely c h r y s o t i l e , which belongs t o t h e s e r p e n t i n e s e r i e s and t h e amphiboles, which i n c l u d e c r o c i d o l i t e , a c t i n o l i t e t r e m o l i t e , amosite and a n t h o p h y l l i t e . The c r y s t a l l i n e s t r u c t u r e s of b o t h c h r y s o t i l e and amphiboles have been c l a r i f i e d . C h r y s o t i l e c o n s i s t s o f a l a y e r of magnesium oxide-hydroxide octahedra bonded t o a layer o f s i l i c o n dioxide tetrahedra. The s h e e t l i k e l a y e r tends t o r o l l i t s e l f i n t o a h o l l o w t u b e w i t h t h e magnesium hydrox-
.
TABLE 10.2 Modified Nucleosides and B-AIBA Content in Serum pmol/ml N $
m1I ml G ac4C m2G m:G t6A 8-AIBA
37 21 18 29 27 19 10 20
X
2810 41.5 41.0 71.6 19.8 47.1 38.2 2140
Ma1 e nmollpmol of creati nine -
U
523 9.80 17.1 18.9 7.59 20.0 12.6 960
X
40.3 0.61 0.61 1.04 0.29 0.73 0.59 32.4
pmol /ml U
8.07 0.16 0.27 0.27 0.12 0.40 0.18 14.7
N
48 35 16 36 33 29 19 27
T
2520 42.8 56.9 61.6 18.9 46.9 32.5 1680
Femal e nmollpmol o f creatinine U
538 17.2 18.4 20.1 5.72 21.4 10.5 684
-
X
46.3 0.76 1.15 1.11 0.35 0.91 0.62 30.7
U
12.5 0.29 0.55 0.35 0.13 0.50 0.23 12.5
D e t a i l s a r e same as f o r Table 9 . 1 . Males 19-56 years and females 21-52 years o l d . C r e a t i n i n e i n serum (males 13.8 nmol/ml) was determined by HPLC ( r e f . 52). 70.8 2 11.1; females 56.5
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ide on the outer surface. In contrast, amphiboles consist of doubl e chains of 1 inked si 1 i con-oxygen tetrahedra posi t i oned para1 1el t o the vertical crystal 1 ographi c axis and bound 1a t e r a l l y by m e t a l l i c ions. The chemical composition of the two asbestos types a l s o d i f f e r i n t h a t chrysotile contains l e s s s i l i c a and iron oxides than the amphiboles, b u t has a higher content of magnesium than the amphiboles ( r e f . 18). Asbestos-contai n i ng materi a1 s have been used extensively i n industry since i t s commercial introduction i n the l a t e 19th Century. I t i s estimated t h a t some 3,000 types of products contain some form of asbestos. One of i t s most important uses has been as a component i n heat and f r o s t insulation, and asbestos i s therefore often encountered i n s h i p b u i l d i n g and r e p a i r , construct i o n , power production, chemical manufacturing and i n the manufact u r i n g of automobile brakes ( r e f . 19). Adverse health e f f e c t s of inhaled asbestos f i b e r s began t o appear among occupational ly-exposed groups a t the e a r l y p a r t of the century. I t was subsequently shown t h a t inhaled asbestos f i b e r s can be retained i n the l u n g t i s s u e , where they can be i d e n t i f i e d e i t h e r by l i g h t microscopy, phase contrast microscopy, and electron microscopy, depending upon f i b e r s i z e and s t r u c t u r e ( r e f s . 20-22) . Asbestosis, i . e . , i n t e r s t i t i a l pulmonary f i b r o s i s , was the f i r s t disease related t o exposure t o airborne a s b e s t o s . ' I t i s s t i l l the most common manifestation of asbestos-induced e f f e c t s among occupational ly-exposed individuals. There i s usually a 1 atency period of approximately ten years before radiographic abnormalities occur. These are characterized radiographically by l i n e a r and r e t i c u l a r opacities affecting the middle and lower l u n g f i e l d s . I t i s also known t h a t a group of conditions of the pleura such a s thickening and c a l c i f i c a t i o n can be induced by asbestos ( r e f . 23).\ The f i r s t indications t h a t asbestos exposure m i g h t r e s u l t i n cancer appeared i n the 1930's, when a few cases of, 1 ung cancer were reported i n persons w i t h asbestosis ( r e f . 2 4 ) . I t was subsequently shown, i n epidemiological s t u d i e s , t h a t populations of individuals w i t h a h i s t o r y of occupational exposure t o asbestos
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have h i g h m o r t a l i t y r a t e s o f l u n g cancer ( r e f . 25-27). I t was a l s o c l a r i f i e d t h a t asbestos seems t o e x e r t i t s e f f e c t s y n e r g i s t i c a l l y w i t h tobacco smoke, and t h a t asbestos i n s u l a t i o n workers who smoke c i g a r e t t e s r u n t h e r i s k o f an e x t r a o r d i n a r i l y h i g h m o r t a l i t y r a t e i n l u n g cancer ( r e f . 28, 29). However, t h e r e i s some e v i dence, based. upon a small number o f d a t a p o i n t s , t h a t asbestos may i n c r e a s e t h e r i s k o f l u n g cancer even i n nonsmoking i n d i v i d u a l s . I t was r e c e n t l y observed t h a t t h e m o r t a l i t y o f l u n g cancer decreased s i g n i f i c a n t l y among asbestos workers who d i s c o n t i n u e c i g a r e t t e smoking as compared t o t h o s e who c o n t i n u e t h i s h a b i t ( r e f . 30). Although l u n g cancer may be t h e most common neoplasm among asbestos workers, m a l i g n a n t mesothel ioma o f t h e p l e u r a and p e r i toneum i s t h e most a s b e s t o s - s p e c i f i c m a l i g n a n t d i s e a s e ( r e f . 31). An e x t r e m e l y r a r e tumor i n t h e general p o p u l a t i o n , i t accounts f o r a p p r o x i m a t e l y 8% o f a l l deaths o f asbestos i n s u l a t i o n workers, a c c o r d i n g t o some s t u d i e s ( r e f . 17). Survival time i s usually l e s s t h a n a y e a r a f t e r d i a g n o s i s w i t h c u r r e n t l y a v a i l a b l e diagnost i c methods and t r e a t m e n t m o d a l i t i e s . I t i s o f i n t e r e s t t o note t h a t c i g a r e t t e smoking does n o t appear t o be r e l a t e d t o t h e r i s k o f devel o p i ng mesothel ioma ( r e f . 29) A c l e a r dose response r e l a t i o n s h i p , which seems t o e x i s t f o r asbestosis, has n o t been i d e n t i f i e d w i t h t h e same degree o f c e r t a i n t y f o r e i t h e r l u n g cancer o r mesothel ioma. I n a d d i t i o n t o l u n g cancer and mesothelioma, o t h e r neoplasms have a1 so been a s s o c i a t e d w i t h o c c u p a t i o n a l exposure t o asbestos. These i n c l u d e c o l on-rectum cancer, esophageal and stomach cancer, as w e l l as cancer o f t h e oropharynx and l a r y n x ( r e f s . 17, 32, 33). I t should be emphasized, however, t h a t t h e degree o f evidence f o r t h e a s s o c i a t i o n between t h e l a t t e r groups o f cancers and asbestosexposure i s l e s s s t r o n g than f o r l u n g cancer and mesothelioma ( r e f . 34). As m e n t i o n e d , a s b e s t o s - r e l a t e d diseases, including the asbestos-associated cancers, usual l y become c l i n i c a l l y mani f e s t o n l y a f t e r a l o n g t i m e l a p s e f r o m onset o f exposure. For asbestosis, t h i s i s u s u a l l y a decade o r so, b u t f o r t h e asbestosr e l a t e d n e o p l a s t i c diseases, two t o f o u r decades i s u s u a l l y t h e r u l e , D u r i n g t h a t l a t e n c y p e r i o d , t h e i n d i v i d u a l may e i t h e r have
.
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radiographic evidence of asbestosis or may be entirely without symptoms or signs of exposure. I n either case, the exposed individual does not manifest any sign or symptom that would make possible the identification of the individual's high neoplastic risk. The protracted period of latency poses a difficult problem for early detection and, especially, for successful therapy. The information obtained in retrospective prospective epidemiological investigations of asbestos insulation workers provides evidence for the serious consequences that may occur when there has been a delay in establishing a cause-effect relationship, and when the biological and biochemical events during the 1 atency period remain poorly understood. Because of this situation, there is an urgent need for developing new diagnostic methods which could assist i n the identification of pathophysiological changes at a stage when early intervention might be of added advantage. We report observations of the urinary excretion patterns of modified nucleosides in patients with malignant mesothel ioma as a possible approach for the early detection of this disease. The first pilot study addressed the question whether patients w.ith asbestos-associated ma1 ignant mesothel ioma excrete elevated levels of modified nucleosides i n their urine. Eight patients with mesothelioma were studied and the results showed that several nucleosides were elevated, with $ elevated in all patients, Table 10.3 (ref. 35). Subsequently, similar results were obtained from additional seven patients with ma1 ignant mesothel ioma (Unpublished observation). These results indicate that mesothelioma produces elevated excretion of nucleosides and that this may be an additional diagnostic tool . No fa1 se-negative resul ts were observed i n this small group of patients with mesothelioma and the laboratory investigators were able to identify all as "cancers" on the basis of the quantitation of nucleosides. A recent stu y has shown increased excretion of $ and hypoxanthine in nude mice transplanted with mesothelioma (ref. 49). Excretion of $ s related to the growth of mesothelioma and an increased excretion commences at a time when the tumor is just measurable. The potentially useful predictive value of study ng nucleo-
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side excretion patterns i s reflected i n the i n i t i a l p i l o t study. Ten of 13 workers whose onset of asbestos exposure preceded the examination date by 30 t o 40 years had elevated levels of nucleosides. Elevated excretion of pb was the p r i n c i p a l a b n o r ma l i t y . This i s of interest since this gr oup of workers constitutes a population a t p a r t i c u l a r l y h i g h risk of neoplastic disease development (ref. 17). Follow-up information t o date on 3 of the 10 workers gave evidence of cancer. One developed lperi toneal mesothe1 i oma. A second study was therefore undertaken t o investigate whether apparently well asbestos insulation workers w i t h l o n g histories of asbestos exposure (30 years or longer), b u t w i t h o u t current evidence of cancer, would show unusual urinary excretion patterns of modified nucleosides. A group of 47 male individuals TABLE 10.3
Elevated Modified Nucleosides i n Patients w i t h Mesothelioma Patient 1 2 3 4 5 6 7 8 Normal
Sex
Marker Levels (nmol/fimol creatini ne) $
M M M M M
49.2 41.1 114.5 38.6 32.9 M 38.4 M 48.3 M 36.6 M 22.4
mlA
1.80 1.84 4.85 2.97 NRa 3.23 2.78 2.22 1.77
PCNR
2.81 1.74 3.75 1.90 1.97 1.90 1.02 1.56 0.87
m'I
mlG
mzG
m:G
B-AIBA
2.30 2.85 5.50 2.16 1.69 1.94 1.63 1.31 1.15
0.87 1.20 3.76 NR 1.44 2.15 1.30 0.79 1.06
0.54 0.44 1.46 0.53 0.73 0.73 0.40 0.68 0.35
2.19 2.26 4.32 1.96 1.72 2.24 1.44 1.30 1.2
6.0 4.0 21.0 30.0 3.0 4.0 9.4 4.0 4.27
(k2.10)b(*0.29) (i0.25) (i0.27) F
26.7
1.76
1.05
1.18
(e4.5) (k0.48) (+0.26)(e0.39) aNR, N o t R e s o l v e d hean S.D.
(k0.07) (20.15) (k1.93)
1.07 0.41
1.44
5.8
(k0. 12) (k0.38) (k4.11)
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was examined. They had been selected from a population of close to 2,000 workers from whom we have collected urine samples in the course of cl i ni cal examinations. The results of this feasi bi 1 i ty study showed that asbestos insulation workers exhibit significantly higher levels of seven of the investigated nucleosides when compared to .b control group. Twenty-seven (57%) of the asbestos workers had three or more nucleosides elevated, indicating a highly abnormal excretion profile (ref. 36). ' An increasing severity of radiographic a1 terati ons was associated with a greater frequency of elevated nucleosides, especially with mrA, mlI, mlG, and m$G. Duration since onset o f exposure was directly related to $, mlI, and m$G. We have also examined d
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Because of the possi bi 1i t y of re1 a t i ng .the nucl e o s i d e l e v e l s t o e x t e n s i v e c l i n i c a l and l a b o r a t o r y information a v a i l a b l e on this high r i s k population, i t i s a n t i c i p a t e d t h a t important information w i l l be obtained about determinants of nucleoside l e v e l s and m u l t i p l e f a c t o r i n t e r a c t i o n s which may g i v e c l u e s t o p r e v e n t i v e measures ( r e f . 38). 10.2.2 Urinary Excretion of Modified Nucleosides i n P a t i e n t s w i t h Various Manifestations o f I n f e c t i o n with HIV The d i s e a s e , Acquired Immune Deficiency Syndrome (AIDS) has r e c e n t l y approached epidemic proportions i n many p a r t s of the world. AIDS i s c h a r a c t e r i z e d by severe immune depression and s u s c e p t i b i l i t y t o neoplasms and o p p o r t u n i s t i c i n f e c t i o n s . Recent evidence s t r o n g l y i m p l i c a t e s a lymphotropic r e t r o v i r u s , HIV, a s the primary e t i o l o g i c agent of AIDS ( r e f . 39-42). During a f i v e y e a r period, 5-19% of i n d i v i d u a l s with HIV a n t i b o d i e s developed AIDS and there was a l a t e n c y period o f 1-5 y e a r s between i n f e c t i o n and development of d i s e a s e . Pathognomonic immunological o r biochemical a b e r r a t i o n s t h a t d e l i n e a t e t h i s syndrome a r e not known. We have observed e a r l ier e l evated 1eve1 s o f modi f i ed nucleosides and j-AIBA i n u r i n e from p a t i e n t s w i t h AIDS and i n a male homosexual population from New York C i t y ( r e f . 43). A goal of o u r s t u d y i s t o explore whether i t i s p o s s i b l e t o i d e n t i f y immunological o r biochemical abnormal i t i e s i n persons with HIV i n f e c t i o n s t h a t may i d e n t i f y s u b j e c t s with p r e d i s p o s i t i o n f o r development of AIDS. Conceivably, such a s t u d y might allow selection of subjects f o r early therapeutic intervention. The s u b j e c t s (20-52 y e a r s o l d ) f a l l i n t o f o u r c a t e g o r i e s : 1) T h i r t y - f i v e h e a l t h y homosexual men (HHS), none of whom a r e c h r o n i c c a r r i e r s of h e p a t i t i s B s u r f a c e a n t i g e n ; a n a l y s i s of s t o r e d frozen serum by an enzyme-linked immunosorbent assay (ELISA) f o r HIV antibody divided t h i s group r e t r o s p e c t i v e l y i n t o two subcategori e s : 20 h e a l t h y homosexual men who were s e r o n e g a t i v e (HHS-) f o r antibody t o the HIV (ELISA r a t i o l e s s than 3.00) a t the time of the immunol ogi c'al and c l i n i cal eval u a t i ons and nucl e o s i de d e t e r minations and 15 heal t h y homosexual men who were , s e r o p o s i t i v e (HHS+) f o r antibody t o the HIV (ELISA r a t i o g r e a t e r than o r equal t o 5.00) a t the time of e v a l u a t i o n s ; 2) 38 asymptomatic homosexual
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men w i t h Chronic lymphadenopathy syndrome (CLS), defined a s d i s c r e t e lymph nodes i n two o r more non-inguinal s i t e s of three o r more months duration ( r e f . 44); none of t h e s e p a t i e n t s had symptoms o r f i n d i n g s of fevers, n i g h t sweats, w e i g h t l o s s , f a t i g u e , d i a r r h e a , Herpes z o s t e r , Herpes simplex o r i d i o p a t h i c thrombocytopenia; 3) 14 s u b j e c t s w i t h AIDS r e l a t e d complex (ARC) defined a s a syndrome of i n t e r m i t t e n t o r continuous fevers >38.50 f o r > one month, watery d i a r r h e a of two weeks d u r a t i o n f o r which no o t h e r e t i o l o g i e s were e s t a b l i s h e d , unexplained weight l o s s of >15 pounds o r >lo% of the body weight o r minor o p p o r t u n i s t i c infect i o n s such a s oral c a n d i d i a s i s o r Herpes z o s t e r ; and 4) 21 homosexual men w i t h AIDS who met epidemiologic s u r v e i l l a n c e d e f i n i t i o n o f the Centers f o r Disease Control. Only one of the ARC p a t i e n t s had noted s i g n i f i c a n t weight l o s s d u r i n g the weeks p r i o r t o the study. Of the p a t i e n t s w i t h AIDS, twelve had o p p o r t u n i s t i c i n f e c t i o n s (01), and nine had Kaposi's sarcoma (KS). The control population consisted of 52 healthy a d u l t heterosexual males ranging i n age from 18-60 y e a r s o l d . Assays f o r a n t i b o d i e s t o HIV were performed on s t o r e d frozen serum samples using ELISA (ref. 4 5 ) . P a t i e n t s w i t h AIDS, CLS, ARC and asymptomatic homosexuals (HHS) excreted increased amounts of markers i n the urine ( r e f . 4 6 ) . The numbers of abnormally excreted markers i n the urine of p a t i e n t s increased by d i s e a s e category ( F i g . 10.1). P a t i e n t s w i t h AIDS had g r e a t e r e x c r e t i o n of nucleosides t h a n any o t h e r group; 74% o f the p a t i e n t s w i t h CLS had a t l e a s t one abnormal marker, and 42% had t h r e e o r more e l e v a t i o n s ( F i g . 10.2). In c o n t r a s t , over 90% of p a t i e n t s w i t h AIDS o r ARC had one abnormality i n nucleoside excretion and over 75% of persons w i t h AIDS o r ARC had abnormal excretion of t h r e e nucleosides. Interestingly, 80% of the HHS had elevated e x c r e t i o n of one o r more markers (Fig. 10.3). HHS t h a t were p o s i t i v e f o r HIV antibody (HHS(+)), e x h i b i t e d a s i g n i f i c a n t l y different nucleoside excretion p a t t e r n than those who were negative f o r HIV antibody Only 30% of the HHS(-) i n d i v i d u a l s excre\ted abnormal (HHS(-)). amounts of the three markers while 60% of HHS (+) s u b j e c t s exc r e t e d abnormal amounts of t h r e e nucleosides. Amongst a l l markers, t 6 A was c o n s i s t e n t l y elevated i n over 70% of the HHS(+)
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F i g u r e 10.1. Abnormal e x c r e t i o n o f m o d i f i e d n u c l e o s i d e s by pat i e n t s w i t h A I D S w i t h K a p o s i ' s sarcoma KS), A I D S w i t h o p p o r t u n i s t i c i n f e c t i o n s (01), persons w i t h t h e A DS-related com l e x (ARC o r t h e g e n e r a l i z e d c h r o n i c lymphadenopathy syndrome ([LS and I V a n t i b o d y - p o s i t i v e asymptomatic homosexual men (HHS+). T e abnormal values r e p r e s e n t c o n c e n t r a t i o n s more t h a n two s t a n d a r d d e v i a t i o n s g r e a t e r t h a n t h e mean f o r t h e heterosexual c o n t r o l s u b j e c t s . Modi f i e d n u c l eosides were determined by HPLC u s i n g a Radi a1 -Pak c a r t r i d g e ( r e f . 10). t3-AIBA ( b o t h f r e e and bound) was analyzed by HPLC u s i n g f l u o r e s c e n c e d e t e c t i o n o f t h e e l u a t e w i t h o - p h t h a l a l dehyde ( r e f . 48). M o d i f i e d n u c l e o s i des and B-AIBA were expressed relative t o creatinine.
I
1,
A
The g r e a t e s t d i f f e r e n c e s and i n 58% o f t h e HHS(-) s u b j e c t s . between s e r o n e g a t i v e and s e r o p o s i t i v e HHS were seen w i t h m'l, ac4C, #, and m$G. There were a l s o d i f f e r e n c e s i n Qe e x t e n t o f e l e v a t i o n o f v a r i o u s markers determined by Z values, between
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0.0
2.0
4.0
6.0
8.0
10.0
NUMBER OF ABNORMALITIES
Figure 10.2. Numbers of abnormalities in excretion of nucleosides amongst members o f various clinical groups. CLS, KS, and 01. greater excretion of mlI, ac4C and m2G, t h a n HHS(-) subjects (Column 1). However, with the exception of m l G , no such positive correlation was evident between HHS(+) subjects and CLS (Column 2 ) . The most striking differences were between the CLS and ARC groups in which ARC-patients excreted greater amounts of seven of the ten urinary markers (Column 3 ) . Whereas there were few differences between ARC subjects and those with AIDS with Kaposi's sarcoma or AIDS with opportunistic infections (Columns 4 a n d 5 ) . In an earlier sbudy, two of the modified nucleosides, $ and mzG, correlated significantly with the number of s i t e s of palpable lymph nodes. The correlation remained significant w h & b o t h
loo
J
z U
75
-
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HETEROSEXUAL &---A HHS (-1 HHS (+I
0
z U
50
I-
z W
0
5 n
25
0
0.0
2.0
4.0
6.0
8.0
10.0
NUMBER OF ABNORMALITIES
F i g u r e 10.3. Comparison o f m o d i f i e d n u c l e o s j d e e x c r e t i o n b y H I V a n t i body-negative (HHS-) and H I V a n t i body-posi t i v e (HHS+ asymptom a t i c homosexual men. Both groups d i f f e r from heal t h y . e t e r o s e x ual c o n t r o l s and t h e HHS+ s u b j e c t s have more a b n o r m - a l i t i e s than HHS- s u b j e c t s .
b
i n g u i n a l and submandi b u l a r lymph nodes were excluded [ r e f . 43). M o d i f i e d nucleosides, $, mlI, m l G , ac4C, m*G, m$G, t 6 A and PAIBA, were a l s o determined i n t h e matched s e r a of s u b j e c t s . In general i n serum, e l e v a t e d e x c r e t i o n o f t h e s e markers s i m i l a r t o t h a t i n u r i n e was observed i n HHS s u b j e c t s and t h o s e w i t h CLS, ARC, o r A I D S . However, when t h e mean values o f markers i n serum i n v a r i o u s d i a g n o s t i c groups were compared, no s i g n i f i c a n t c o r r e l a t i o n between serum markers and HHS s u b j e c t s and s u b j e c t s w i t h CLS and t h o s e w i t h ARC was e v i d e n t . Therefore, e x c r e t i o n o f markers i n u r i n e appear t o be more r e l e v a n t t h a n serum f o r t h e s e studies. The e f f e c t o f c h r o n i c v i r a l i n f e c t i o n s and use o f i n t r a v e n o u s drugs on t h e e x c r e t i o n o f v a r i o u s m o d i f i e d n u c l e o s i d e s has n o t been s t u d i e d i n d e t a i l . Chick e m b r y o f i b r o b l a s t s i n f e c t e d w i t h
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TABLE 10.4 C o r r e l a t i o n Between Markers and D i a g n o s t i c Groups (Ref. 43) \
H I V (-) HIV,(+) $
mlA PCNR m1 I m1 G ac4C mzG m$G t6A j-AIBA
NSa NS NS p=O. 005 NS p=0.02 p=O. 04 NS NS NS
VS.
D i a a n o s t i c Grouos HHS(+) VS. CLS V S . c LS ARC NS NS NS NS p=O. 024 NS NS NS NS NS
p=o. 02 NS p=o. 001 p=o. 002 NS p=O. 007 p=o. 02 p=O.Ol p=o. 0 1
NS
ARC V S . KS
ARC 01
NS NS NS NS NS NS
NS NS NS p=O. 04 NS NS NS NS NS NS
NS NS NS NS
VS.
aNS, No significant correlation ( p > 0.05).
Rous sarcoma v i r u s e x c r e t e e l e v a t e d l e v e l s o f $ ( r e f . 47). We have observed t h a t i n f o u r a d u l t male s u b j e c t s p o s i t i v e f o r h e p a t i t i s B antigen, o n l y one s u b j e c t e x c r e t e d e l e v a t e d l e v e l s o f markers. I n d i v i d u a l s a t h i g h r i s k f o r AIDS, u s e r s o f m a r i j u a n a and cocaine had s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s between a c u m u l a t i v e i n d e x o f o v e r - a l l drug use and e x c r e t i o n o f PCNR and m2G. Users o f amyl n i t r i t e and e t h y l c h l o r i d e e x c r e t e d l o w e r l e v e l s o f markers ( r e f . 43). I n a recent study i n c o l l a b o r a t i o n w i t h D r . Joyce Wallace ( r e f . 50), we have found t h a t asymptoma i c women i n a methadone maintenance program from New York C i Y I p o s i t i v e f o r H I V antibody, e x c r e t e d e l e v a t e d l e v e l s o f m o d i f ed nucleosides compared t o women who were n e g a t i v e f o r a n t i b o d y t o HIV. The mechanisms t h a t produce abnormal n u c l e o s i d e e x c r e t i o n i n asymptomatic a d u l t s and i n s u b j e c t s w i t h H I V d i s e a s e a r e unknown. A p r o s p e c t i v e s t u d y i s under progress t o a s c e r t a i n t h e u s e f u l n e s s o f measuring m o d i f i e d n u c l e o s i d e e x c r e t i o n t o i d e n t i f y s u b j e c t s who w i l l progress t o ARC o r A I D S and t o m o n i t o r response t o treatment.
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10.3
1.
2.
3.
4.
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