Mutagenicity in urine from nurses handling cytostatic agents

Mutagenicity Cytostatic SIMONE

in Urine

de Rccherches

FRANCOISE

CALLAIS,t

en Epidhmiologie

Institut Gustaoe Rousv, :D$artement

Handling

Agents

BENHAMOU,*

‘l’niti

from Nurses

de Statistique

des Cancers de l’lnstitut

Villej,if;

.2lddicale,

HfiLI?NE

tLaboratoire

SANCHO-GARNER,: 11 COURTOIS$

,vational

d’Hygiine,

SRENG MIN,t$ YVES and BERSARD FESTY+$

de la Santi et de la Recherche

de Pru’uention et &Education

Institut Gustave Rous.~y, l’illeju$

and SLaboratoire

Sanitaire,

d’&giine

.Wdicale

(U287),

InicersitP

Parir

de la Wle

de Paris.

I-:

Paris, France

Abstract-J

cohort stud]) of 29 nurses Lelho extensively handle cytostatic drugs, 29 controls

matched on sex and age, and seven patients and 1985. In a ,first study, urinav

Salmonella

was carried out between

assays performed

for nurses as compared to controls, after adjustment on smoking

The results of mutagenicify assays for patients were sign$cantly

,f0r non-smoker

nurses and non-smoker

(P < 0.05)

non-smokers,

that is 1 1 nurses and

ulho handled at least one electrophilic drugs towards

was sign$cant~

increased

agents as compared to nuws

in animals.

negative

drugs, widely used in cancer have been shown to be carcinogenic

mutagenicity

Some

agents

of thcsc

have

tumors

drugs,

been

particularly

associated

in patients

with

under

studies

ihlc health

howevrr,

chromatid to

this

mosome rcsportcd qurncy,

has

finding

These

increased

among

[ 61. An increase but

not

nurses

[ 31. Morcovcr,

findings

study

comparing

nurses

who

reported

and

prompted urinary

about

exposure

urinary to cyto-

us to perform mutagenicity

extensively

therapeutic

con-

agents,

non-exposed

as com[2,

handle

patients

a cohort

in oncology

cancer

under

chemo-

chrmothcrapy

controls.

aberrant

of chrorcccntl) gaps

to

frcand

hcceptcsd

22 May 1986. .\ddrcss for reprints and correspondence: Simone Institut Gustave Roussy, D6partemcnt de Statistiqur Rur Camilk Dcsmoulins. 91805 \‘ilkjuif. France. Supported by thr Institut Gustave Roussy Contract At)hrcviation used: CI =
AND

No 83 112.

1489

Gustave

was car-

Roussy

from

1983

nurses

(28 women

and

two

men,

mean

age 31.6 yr) working in oncology participate in the study. Of them,

units agrrcd to 10 wcrc smokers.

The

drugs

nurses

noted

the

routinely

handling.

injcctablc admixed

antincoplastic by week and

This

Benhamou. S16dicak.

METHODS

exposed-non-exposed,

1985. Thirty

frrqucnc) positive

study,

ried out at the Institut

lym-

frequency

breaks

i\ cohort

[a,

has

been

MATERIALS

31;

confirmed

both

been

occupational

frcqucncy

in chromosome

not in chromosome

\vas also rcportcd

with

reported been

poss-

to small

in nurses

chromosomally

a significant breaks

been

has

drugs, increased

exchanges

controls

I, 1. Concerning phocytrs,

therapeutic

,4 significantly

about

cxposcd

have and

[13].

second

chemotherapy

performed

in personnel

of cancer results.

of sistc.r pared

ha\.c brcn

hazards

results

static agents [4, 5, 7-121. The results of thcsc different studies arc summarized in a rcccnt paper

alky-

111. amounts

in nur.rer

who handled non-electrophilir

INTRODUCTION chemotherapy,

Scvcral

TA 98 with and

(P < 0.05)

T.4 98 with and u:ithout S9 mix.

MANY cytostatic

flicting

1I matched controls,

was detected for nurses as compared to matched controls towards

malignant

(P < 0.01) than

TX 100 and T.4 I.535 ? S9 mix. rl signiJicant increase in mutagenic acti@

without S9 mix. LMoreover. the mutagenicily

lating

higher

controls with Trl 98 without S9 mix. Of the 29 pairs,

complementary assays ujere pcrformedfor with TA 98,

1983

re,ith the rlnles test tozr,ards

Trl 98 with and without S9 mix gave an increased mutagenic

typhimurium

activity, although not sign$cant, habits.

under chemotherapy

mutagenicify

number

different

and

for each

and

admixed

and

the

mean

duration

wore

no gloves

or other

they

were

numbrr

of

agents prcparcd and the duration of cxposurr.

of preparations

prepared

the

of antitumor

was in average of cxposurc protcctivc

drugs

65 by wrck, -1- yr.

clothing,

They cxccpt

1490

S. Benhamou

seven nurses who handled methotrexate with gloves. The drugs most frequently handled include 5 fluoro-uracil, cyclophosphamide, vincristine, doxorubicin, vindesine, cisplatin and dacarbazine. A 24-hr urine sample was collected for each nurse after a 3-day work shift, during an off-duty day, from Saturday morning until Sunday morning. Thirty office clerks of the hospital, matched by sex and age (+ 3 yr) were included. Seven were smokers and none had ever had any contact with cytostatic drugs. The 24-hr urine samples were collected after a working week. Each exposed and non-exposed subject was interviewed on possible exposure to other mutagenic agents. The questionnaire included questions about X-rays, regular medications over the last 5 yr, illnesses over the last 6 months, oral contraceptives, coffee, and hair dyeing in addition to smoking. No significant difference of exposure to such agents was found between nurses and controls. A positive control group consisting of seven nonsmoker patients undergoing intensive chemotherapy (principally vincristine, cyclophosphamide, vindesine, bleomycin) at the hospital was included in the study. A 24-hr urine sample was also collected from each of them. Urine treatment and mutagenicity assays

Each 24-hr urine sample was homogenized. A sample was drawn in order to determine the crcatinine level. According to the volume of urine available, for each subject, one to three samples of 200 ml of urine were brought to pH 7 with NaOH lN, filtered through Whatman No 1 filter paper and treated with the method of Yamasaki and Ames [ 141. After passage of each urine sample through XAD-2 resin (flow rate 2-3 ml/min), the resin was washed with 10 ml of distilled water and eluted with 10 ml of acetone. Each cluate was evaporated to dryness at 40” C under nitrogen, and the residue was taken up in 0.8 ml of dimethylsulfoxide and stored at -20” C. Mutagenicity assays were carried out according to Maron and Ames’s method [ 151, using Salmonella typhimurium strain TA 98, with and without a mctabolic activation system S9 mix (containing per ml cofactors and 50 pl of S9 prepared from livers of adult male Sprague-Dawley rats pretreated with Arocolor 1254). Histidine has not been observed in urine concentrates in high enough amounts to induce bacterial growth in the test system. Morcover, no mutagenic activity was detected with blanks performed with distilled water and treated in the same conditions as urine. For each assay, 50, 100 and 200 ~1 urine concentrates (corresponding to 12.5, 25.0 and 50.0 ml of urine)

et al. were used, and assays were performed twice for each subject. These doses were reduced when cytotoxicity appeared. For patients, lower samples of urine concentrates, as little as 5-50 p.1, were used because of the cytotoxicity at 100 and 200 ~1. After 2 days in an incubator at 37” C, the revertants were counted without knowledge of cytostatic exposure. A complementary study was performed only in 11 pairs of non-smokers still working at the Institut Gustave Roussy. Twenty-four-hour urine samples were collected again for nurses after 3 days of exposure to cytostatics, and during a day of work. Each day, the name of cytostatics handled, and for each the number of injectable antineoplastic drugs prepared and administered were collected. The urine treatment was identical to the one of the first study, and mutagenicity assays were performed with Salmonella pphimurium TA 98, TA 100 and TA 1535 with and without S9 mix. For each strain, three or more urine concentrates were used, and assays were also performed twice for each subject. Statistical

analysis

The data were analysed by BMDP programs (Biomedical Computer Programs, 198 1). The slope of the linear regression was calculated from the two assays for each subject. A positive mutagenic activity was defined when a significant correlation (at 0.05 significance level) between number of His+ revertants and volume of urine was observed, and otherwise the result was considered negative. The number of induced His+ revcrtants/ g of creatinine was calculated as: His+/g creatinine = His+/ml

urine * Volume g of creatinine/24

urine/24

hr.

hr

Mutagenicity was expressed as number of His+/g creatinine for positive urine concentrates, and the value 0 was assigned to negative ones, The urine mutagenicity results were compared using two-way analysis of variance taking smoking status into account (ANOVA program) after a logarithmic transformation of data, or using a permutation test

[161. RESULTS One nurse was unexposed at the time of urine collection, so the pair (exposed and matched nonexposed) has been excluded. The results of urine mutagenicity arc presented in Table 1 for each group of subjects defined. Using a two-way analysis of variance, the geometric mean of His+ revertants/g creatininc towards TA 98 without S9 mix, was found increased in nurses (2.83, 95% CI : 1.78-4.50) as compared to matched controls

Antineoplastic Table 1.

Agents, Nurses,

Number of His’ revertants/g creatinine among nurses, matched controls and patients with TA 98 - S9 mix Nurses Controls

Nonsmoker controls

nurses

and

Smoker nurses and nonsmoker controls

nurses controls

Nonsmokrr

smoker Smoker controls

nurses

and

and

+ S9 mix Nurses Controls

257 0 0 269 168 208 0 0 0 170 522 297 0 0 0 0 0

215 0

463 2X 0 0 0 0 0 0

743 0 8899 0 716 612 3016 228 648 275 632 .j 13 .X9 0 0

820 840 836 0 0 0 0 863 290 414 341 86i 0 ,576 1452

0 198

0 0

286 0

470 395 0 0 0

0 0 0 0 0

10260 1999 I 6671 3428 321.5

0 959 0 0 57 1

316 0

0 0

0 0

11570 2408

1427 285 0 0 0

0 0 65.3 0 0

9211 2733 34.57 2644 15.5 i

1110 490 s297 14271 0

I80 0 0 0 0

Patients 13152 4092 l-483 0 0 0 0

(1.66,

95%

98 with

CI : 1.15-2.41).

Sg’mix,

(7.25,

95% were

smoking

habits.

geometric

not

compared

significant With

mean

atininc

with

higher

(P c 0.0001)

95%

as

TA

respect

in

was in

difl on

habits, of crc-

those

(29.90, of

Paticnta 28224 5.55 1 381-l 721 0 0 0

patients

than

and

than

lar

results

S9 mix

non-

were

observed

The

results nurses

of urine

and

2. No mutagenic

‘I’,4 1535 with

and

contrary,

between

significantly

higher

Using

a permutation

samples,

the mutagenic

98 without

S9 mix,

test

for two

activity, was

independent

towards

significantly

with

TA

higher

for

100 without

Simi-

98 with

to non-smoker

collected

controls activity

without S9 mix the

pairs.

With

and

a day in

or

activity

in nurses

towards TA a permutation to TX

with

towards

in nurses

mutagenic

respect

11 non-

was dctrctcd

S9 mix

(P < 0.05)

in

during

arc presented

cithcr

pared to matched controls and without S9 mix, using matched

TA

mutagenicity

sample

11 matched

controls. On thr

non-smokers.

towards

as compared

(urine

smokers (5.27, 95% CI : 2.6GlO.45) after adjustmcnt on cytostatic exposure. On the contrary, no difference was found with TA 98 without S9 mix and

(P < O.Ol),

(P < 0.002).

(P < 0.01).

TA

smokers

nurses

controls

for patients

smoker Table

for non-smoker

non-smoker

controls

of work)

significantly

of smokers

than

controls the

to smoking

urine

TA

(10.62,

adjustment

revcrtants/g

S9 mix

CI : 22.26-40.34)

to

However, after

of His+

98 +

with

for nurses

CI : 4.18-12.58).

fcrcnccs the

Similarly,

it was increased

Cl : 6.03-18.73)

95%

1491

Urine Matagenici&

in was

as com98 with test for

100 plus

S9

S. Benhamou

1492 Table 2.

et al.

Number of His+ revertants/g creatinine in non-smoker

nurses and matched controls with

TA 98 and TA 100 TA 98 Nurses

S9 mix Controls

2399 0 0 0 349 0 0 0 1227 4827 1510

the

mix,

3014 0 4060 505 0 0 0 0 1488 4047 1661

215 0 180 0 0 0 0 463 250 0 0 0.05

P value

mutagenic

although

TA 98 + S9 mix Nurses Controls

activity

not significantly,

was

in nurses

drugs, urine

to compare

according

nection

increased,

the

agents

collection,

electrophilic

were

increase

seven

in number nurses

pared

to nurses

TA

drugs,

cytostatic.

among towards

TA

98 with

by nurses

S9 mix

day

drugs at least

3 shows

a significant

to

(P < 0.05)

and

(P < 0.01).

towards

effect

suggested on

Salmonella

that

mutagenic

smoking

Table 3.

habits,

TA

in 100

in

genicity

among

smoking

smoking

controls

could

smoking

con-

in nurses mutagens could

handling

electrophilic

Nurses drugs

handling

non eiectrophilic

P-value

controls,

greater

day

TA 98 + S9 mix

TA 100 + S9 mix

2399 0 0 0 1227 4827 1510

3014 4060 0 0 1488 4047 1661

8754 1336 0 0 0 0 0

0 0 349 0

0 505 0 0

0 0 0 1870

0.02

NS

not

number differ

in controls strain

TA

sig-

( 15.3).

as a consequence with

to

by a dif-

the mean did

and

in urine

muta-

as compared

since

be detected

TA 98 S9 mix

0.05

increased to

The

(15.5)

handled among nurses with TA 98 and TA 100

Nurses drugs

nurses

So, according

exhibited

Number of His+ revertants/g creatinine according to cytostatic drugs

-

of smoking

not be explained per

in

of non-

nurses

behaviour smoked

The

using muta-

in those

compared

significantly.

Moreover,

urine

nurses as

difference

mutagenicity

than controls.

not

smoking

results

in urine

of smoking

activity

no

TA 98 in the presence

nurses and

as com-

controls.

greater

although

nificantly

has a potcn-

activity

pphimurium

controls,

in those

of cigarettes

smoking

strain

nurses

and

different

is, both

to cytostatic a significantly

and

nurses

of non-smoking

ferent It has been

controls,

with

that

mutagenic

drugs

reported in smoking

reported

assays

smoking than

as com-

nonclectrophilic S9 mix

one

crcatinine

drugs

smoking

study

urine

exposure

author

non-smoking

of S9 mix:

[ 171.

handled rcvertants/g

to

genicity

cispla-

DISCUSSION tiating

The

mutagenicity

present

of

have

handling

pared

in two categories:

electrophilic

98 without

the

occupational

[ll].

between

Table

of His+

handling

muta-

nitrosoureas,

C) or non-elcctrophilic

11 nurses,

clectrophiljF

of urine

of antineoplastic

classified

(alkylating

tin, mitomycin Of the

handled

1870 0 0 0 0 0

with

drugs

as compared

the results

to the nature

0 0 784 0 0 0 0 2253 1022 302 0 NS

increased

In order

8754 0 1336 0 0

820 840 836 0 0 0 0 863 290 414 341 0.04

to controls. genicity

TA 100 + S9 mix Nurses Controls

of 98 in

Agents, Nurses,

Antineoplastic the presence from

of S9 mix: mutagenic

smokers

was

non-smokers. those

These

previously

98 plus

results

which

genicity

by anticancer

the smoking

factor

own ef%ect of anticancer Optimal sidered

timing

drugs. revealed

later

Grst

on with

same

way

da)s,

with

first study,

were taking

sometimes

inconsistent

24-hr

samples

4.

5. 6. 7. 8. 9.

IO. II.

12. 13. I %.

15. 16. 17.

the a

presented

significant

according

of Falck

handled.

These

collected

of antineoplastic ard

after

significantly

results drugs

and

without

that

can constitute

muta-

cytostatic

manipulation a hralth

haz-

in nurses.

undertaken in the

of several

results.

wcrc collected

Agency

nurses

urine of

con-

S9 mix.

among in

nature

suggest

incrcasrd

to matched

results the

collection

a day of work)

difl’ercncc to

collection.

urine

as compared 98 with

pcrplacr

conditions

In our after

Acknowledgements-The authors \vish IO thank all the volunterrs who provided urine samples. l>r 11. (;out li)r her contribution to the study, ;Llrs C. Paolctti liu arranging urine collection. and Dr B.N. Ames who provided txwcrial strain\.

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muta-

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mutagenicity

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a strong

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haps

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findings

a greater

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The

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The

could

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some

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could

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as compared

[14];

is the

caused

activity

higher

reported

S9 mix

mutagenicity results

&fold

Urine Matagenicio

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