Inhibition Steven

of nitrosamine

R Tannenbaum,

ABSTRACt’

Nitrosation

conditions large vivo

John

by reaction

phages

and

endothelial

by mammalian NO followed amines

occurs

cells.

The

are synthesized

in the body.

Precursors

constantly

present

almost conditions concerning

action

interest

in vivo and possible

endogenous

formation

nitrosation

inhibitors,

acid, which reacts virtually nontoxic. ascorbic Nuir

acid

most

attention

inhibition

formation to

favorable

has

re-

considerable arising from

ofcompounds.

Among focused

on

many ascorbic

many nitrosating agents and which presentation discusses the chemistry of nitrosation

is of

Am J C/in

reactions.

Sander

and

199 1;53:247S-50S.

WORDS

ascorbic

Nitrite,

macrophages,

nitroso

compounds,

Many

in humans

and

analysis

Much

has been

written

on the endogenous

synthesis

nitrite, and N-nitroso compounds, and the with this subject are a tribute to the current

endogenous

low

diet

nitrate

research.

This

as the

paper

personal

comment

in the field. The exposure multiple acid

not

ofone

nitrosation

long-time

from

valuable

for

some

quantities nitrosation

not give an accurate in various tissues

nogenesis acid agents

occurs in the

to nitrosation

but

measured in urine. The Ohshima and

pathways

but

may

in specific

related may

play

in the

Am J C/in Nutr

tissues.

to endogenous in modulating

is that picture and body

a nitrate-nitrite

balance

of the potential compartments.

for NLarge

This

paper

nitrosation the

examines and

effects

some

the role that of various

of the

Printed

in USA.

© 1991 American

Downloaded from https://academic.oup.com/ajcn/article-abstract/53/1/247S/4691309 by guest on 01 April 2018

All studies

has been

have

when

A molar

acid

[assuming

doses

and

5% ofthe account was

from studies 2-4 nmol/d other

the

acid,

of acid-catalyzed Two possible

CDL 3

acid,

by The National

supported Address

reprint

for Clinical

whereas

likely

are

Institute Institutes

NPRO where was

excretion

has been

obtained

NPRO excretion is be formed at some

one

that

is inaccessible other

nitrosating

than

agents.

nitrogen

of Technology, ofHealth,

(8)] do not

baseline

via a mechanism

atmospheric

yet, nitrite

In studies formation

evidence

background NPRO may

most

probably

the Massachusetts

Supported

of Technology, 02139. Society

stomach,

nitrate,

gastric

to nitrite

nitrosation. sources ofendogenous

to dietary From

the estimated

NPRO. ‘5N-NPRO

(2: 1) is

in vitro,

ofpneformed

Similar

NPRO when a with the

to nitrite

formation

sources

by ascorbic

and

that

is reduced

in the ferret where (9). Consequently, than

and

undoses

nmol/d along

dose

was unaffected.

formed that

Dietary

for the excess urinary given to the subjects,

inhibited

of ‘4N-NPRO

2

nitrate NPRO.

than

by other

i-proline

acid

NPRO

larger

used are

are given

of ascorbic

inhibit

20-fold

urinary

completely

dition

ratio

in a 24-h

demonstrated

nitrate

of a

NPRO is not quantitatively

nitrosamines

respectively)

to completely

ascorbic

site

4 mmol,

dose.

sufficient

ascorbic

nitrosating

(I)

that

increase

because can be

returns to baseline levels of 14-30 (1 g) of ascorbic acid is administered

to ascorbic

body. 1991;53:247S-SOS.

(2, 5-7). levels

method

confirmed

oral doses consuming

is measured

5 and

through mediated

NPRO

de-

in humans.

sequential to a subject

excretion large dose

do not ‘5N-NO to

resulting

Bartsch

have

however,

for demonstrat-

of N-nitrosoproline

(typically

worker

use of N-nitrosoproline nitrosation ( 1 ) has proven

of these

who

NPRO

eliminate

lung

the

to ensure procedures

(1),

method

of this technique, are administered

and

controls

Bartsch

simple

in

of inadequate

collection

urine collection. This method is effective metabolized, is not carcinogenic, and

of nitrite may have only a small contribution to Nif conditions are unfavorable for a reaction. Consmall quantities may play an important role in carci-

versely

issues

field, and

reactions

in the stomach The

ofthe

observer agents

NO2

or bacteria. of endogenous

be universally indicative. What is increasingly apparent

sheet does nitrosation

as a review

to nitrosating

ranging

by mammalian cells (NPRO) as an index to be especially

intended

of people

pathways

catalyzed

is not

dealing area of

and

relatively

formation

example proline

of proper

reaction

nitrosation

because

during

Ohshima

and

In a typical nitrate and

proline

of nitrate,

many papers vigor ofthis

(4).

an effective

ing the

lack

of artifacts

the

could occur in laboratory

endogenous

primarily

(3) and

formation

that

and nitrite nitrosamines

to demonstrate

inconclusive,

techniques

during

signed

attempts

were the

nary

Introduction

demonstrated

animals.

endogenously

acid

(2) first

ingested secondary amines could produce carcinogenic

researchers

KEY

Buerkle

between vivo and

against

compounds

under

of nitrosation

analytical

eg, dimethylamine,

ofN-nitroso

Chemistry

of a

of arginine . Nitrosatable

there is, consequently, human health risks class

with This

some,

together

ofthis

of

any

ofnitrite

oxidation and N204

and

are thus

variety

with

acid

D Leaf

Nitrite can be formed in and by activated macro-

mechanism

foods

by ascorbic

Cynthia

a wide

of amines

enzymatic to N203

in many

and

under

types

species. of nitrate

cells is via by oxidation

are found

S Wishnok,

of most

number of nitrosating via bacterial reduction

formation

oxides

Cambridge,

that in adand MA.

PHS grant CA26731.

by NIH grant T32 ES07020. requests to SR Tannenbaum, Massachusetts Institute 77 Massachusetts Avenue, 56-31 1, Cambridge. MA

Nutrition

2475

2485

TANNENBAUM

nitric

oxide

produced

labile which

species exists

that can react in equilibrium

N2O3

and

N204.

secondary

action

Subsequent

nitrosamines;

water

would

yield

nitrite

of atmospheric (NO2).

possible

nificant

source ppm

1000

50

times

with

to mice

followed

than

Mirvish

et al ( I 1) found

increased

(NDMA)

in the

urine.

positive

correlation

between

levels

in vivo

inhalation

NO2

or nitrosation

of NDMA.

tween

urinary

NPRO

could

indicate

that

inhalation

There and the

of higher

urinary

levels

pathway

and

and

of preformed

was

NDMA

a result

in the

nitrosation

exercise

of

air

or

period

their

etiology association

role

has

be linked

trosation accompany facilitated

eg, a human subject on a nitrate increase in urinary nitrate excretion

the

presence

and

a suitable

nonspecific

intestinal

(LPS)

diarrhea

and

co/i Lipopolysacchande urinary nitrate levels.

fever

(17).

endotoxin Two other

neductase

lines,

when

nitrate

stimulated

and

oxide growth

nitrite

with

trophils

and/or

macrophage

ofL-arginine

(24),

L-arginine

and

brain

in vitro.

This

species lines and We have demonstrated

dogenously

synthesized

Following

administration

arginine, surable

(22).

cells

(25)

produce

production are

added to the corre-

cells

produce

is a general

of nitric

nitric

(23), oxide

phenomenon

in rats,

ferrets,

and

of an intraperitoneal

under

anaerobic

conditions.

forming

LPS-induced

methemoglobin immunostimulation

excreted

nitrate

of ‘5N from

in the

LPS-induced

likely

attributable

In a similar

study,

along

with

‘5N2-L-arginine increases to individual Wagner

and

nitrate

accompanied

a parallel into

in excreted

nitrate

variations et al (I 5) treated

occurs

in humans

and

bacteria

conditions

produce

which

may

nitric

from

in the

have demonstrated synthesized nitrate

L-arginine

to nitrate reactions,

dogenously

could

secondary vivo.

amines

reaction

nitrite only

animals occurs

(37),

by after

have

production

are

clearly

intragastrically

in vitro

bacteria has the exception

is

air

agents

site. Several cells, neutrophils, in the

co/i

E.

types of brain

under

whole

certain

animal.

Ni-

been demonstrated ofbacteria, utilize of nitric

oxide.

We

that L-arginine is a nitrogen source for bioin rats, ferrets, and humans. These same nitric cells may

of L-arginine for nitrosation

The

and

oxide

be duplicated

nitrite

Hollocher

nitrosating

nitrosation

in

when

the

and in at least one other extragastric including macrophages, endothelial

cells,

to nitrite act directly is linked to

(34).

Nitrosation

endogenous

niwhich bacteria

react

be responsible

for this novel

oxidation

in humans. Based on known chemistry it is possible that NO produced enwith

oxygen

to produce

and,

subsequently,

carcinogenic

nitrosate

N-nitrosamines

in

In the by in-

Chemistry

of ascorbic

acid

in incorwide

in rats

in response

studies that

media

that

the

eg, that the and gastric

products

from

nitrogen

(26).

(27).

The

(38) cells

summary,

oxide-producing

increase

nitrate.

In

nitrosation

anaerobically

grown

by Ji and

indicating N2O4.

in elu-

regarding

nitrate

is produced

neu-

of ‘5N2-L-

was

to the system, likely N2O3 and

NO

is the

investigation.

that bacteria this activity

to the

elucidated

from

rats and ferrets excreted ‘5N-NO . Nitrite is not meain mammalian urine because it reacts rapidly with oxy-

hemoglobin

added

and with

of en-

humans dose

are

further

by macrophages All of these cells,

occurring

in cells from many sources. that L-arginine is the precursor nitrate

amine

trosation in vitro.

media (2 1). The is the terminal

Endothelial

also

cell

lymphokines,

via intermediate

can be detected in the for these compounds

across

poration

established

( 19. 20). When suitable secondary amines media containing stimulated macrophages.

guanido-nitrogen

creased

LPS

in vitro

sponding nitrosamines major nitrogen source

rat,

several

This

of endogenous

reducing

nitrosation

did fifth

for 6 h. An urine col-

of mediating

co/i

E.

form

that

demonstrated

and

(34-36).

demonstrated

rophage.

Macrophages

genes

was

by

con-

and

increased Again,

in hypotheses

primarily

who added most

I 2-h

for further

levels

nitrate

on the

exercised.

studies have shown (3 1-33) and that

of nitrate

mechanism

immunostimulants, carrageenan and turpentine, caused smaller, although still significant. increases in urinary nitrate in rats (17). Stuehr and MarIetta ( 1 8) determined that the cell primarily responsible for immunostimulated nitrate synthesis is the mac-

a contri-

days

except

subjects

to increased

or in-

males

cancers. It has been proposed, a higher risk of gastric cancer

nitrosation

synthesis: a ninefold

experiencing

make

been intentionally dosing with nitrate. capable

convert

in vasodilation

healthy

themselves

considered

be due

dogenous balance

treated with Eseherichia had similarly augmented

activated

on excreted

two

remains

mag-

the higher populations of gastric bacteria elevated gastric pH. It was first thought that

(29, 30). Subsequent in amine nitrosation nitrate

a role

for 8 consecutive

cell type

that

the

directly

therefore,

study,

the has than

involved

been

may via

of nitrate, a mammalian process 1 mmol nitrate/d in man under Immunostimulation increases en-

Rats

which nitrate other

of several between

achlonhydria

similarly

or bicycled almost continuously nitrate was observed in the

are another

synthesis to produce (7, 16).

while

exert

during

to the endogenous ( 1 3-1 5) estimated normal conditions nitrate study had

physically

directly

nitrate. ofexercise

In this diet

are will

eg, play

nitrate

found

with

be stimulated

could,

low-nitrate

the cell types

or at a difmay

nor

Bacteria and

also

and

(28).

first time excreted humans by a means cidating

This

examined

correlated which

cells,

of fever

day, when they ran increase in excreted

be-

may

Endothelial

a defined

lection

subsequent

mechanism

for endogenous

NDMA

concentration.

been

sumed

a

oxide

types

to the increase in excreted effects ofa prolonged period

have not

however,

increase

that proline is nitrosated via a different ferent site than dimethylamine. Another

air

NO2

NDMA

20-

not be determined nitrosation of di-

no correlation,

atmospheric

bution The

reported

levels

in the

was

as a consequence

of N-nitrosodimeth-

in human subjects. although it could the increase was due to additional

methylamine

at levels

et al (12)

atmospheric

an-

by LPS.

cell

in urinary They

Macrophages

other

to nitric

increase individuals.

nitrate

induction.

although

sig-

concentrations,

Garland

of fever

by LPS,

a ninefold among

of excreted

directly

of di-

observed variations

enhancement

nitude

as much

to NO2

atmospheric

ylamine excretion whether

contains

administration

by exposure

normal

one

and

large

ni-

represents eg, are

smoke

LPS

and

the

re-

particularly

amines

After

rats with

L-anginine

Cigarettes,

(10).

AL

levels

with

competing

oxides,

cigarette

oxides

is a NO2, agents

nitrate.

endogenous

nitrogen

higher

and

pathway.

of exposure;

methylamine

oxide

compounds the

nitrogen

nitrosation

as

Nitric

ofthese

yield

dioxide

other

reaction

would

reaction

trogen

by cells.

rapidly with oxygen yielding with the potent nitrosating

amines with

The

endogenously

ET

range is most

to LPS.

Sprague-Dawley

Downloaded from https://academic.oup.com/ajcn/article-abstract/53/1/247S/4691309 by guest on 01 April 2018

Some relevant aspects greater detail in earlier here.

Ascorbic

react

with

N2O3

acid, ,

of this topic have been discussed reports but a summary is appropriate

under

H2NO

anaerobic ,

and

NOX

conditions, with

rates

can higher

in

usually in each

ASCORBIC case

than

alkyl

the

in vitro has

corresponding

amines.

nitrosation

been

and

to some

acid

acid

for amides

generally This

in vivo

nitrosation.

to have

potential

scavenger.

This

potential,

importance

realized; not only are the reactions more complex than might have

but the in vitro

model

equilibria,

react

with

amines. can

ascorbic Under

exhaust

the

react

capable

systems acid

NO

are not straightforward.

NO,

to form

does

these

ofthe N203

species

which

conditions capacity

yet to

that occur in been expected,

nitrosating

to form

anaerobic

nitrosating with

several

as

has

reactions

system.

can

nitrosate can

Oxygen,

N2O4 , both

and

that

not

then

however,

of which

are

of nitrosation.

Under

these

removed

circumstances,

from

concentration cesses, ascorbic

the

of nitrosating acid itself

conversion now exists

nitrosation

reactions

inhibiting

gastritis processes

been fully evaluated to the availability partments

of the

acid

significantly

species. can also

In addition react directly

in people. patients

(42).

The

This

has

(41) and role

that

of endogenous but future of reduced human

may

without

to dehydroascorbate that ascorbic acid

for gastrectomy

atrophic

the ascorbic

system

undergoing Evidence strated

themselves

eg, involve

in foods,

however,

be completely live organisms The

the

property

formation

appears

or di-

inhibit

ofcompounds.

nitrosamine

to inhibit

thus

nitrite

rates

therefore

classes

to prevent

extent,

Ascorbic

can

ofthese

exploited

an in vivo

nitrosation

Ascorbic

ACID

body

be effectively affecting

(39, 40). is a limiting recently

carcinogenesis

factor

been

for patients

ascorbic

the

to these prowith oxygen,

with

acid

in

demonchronic

may

play

has

not

in yet

studies should pay close attention ascorbic acid in various cornin populations

at high

risk

for

cancer.

13

References 1. Ohshima H, Bartsch H. Quantitative estimation ofendogenous nitrosation in humans by monitoring N-nitrosoproline excretion in the urine. Cancer Res 198 l;4l:3658-66. 2. Sander I, Buerkle G. Induction of malignant tumors in rats by simultaneous feeding of nitrite and secondary amines. Z Krebsforsch l969;73:54-66. 3. Garland WA, Holowarchenko H, Kuenzig W, Norkus EP, Conney AH. A high resolution mass spectrometry assay for N-nitrosodimethylamine. In: Magee P. ed. Banbury report 12: nitrosamines and human cancer. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories, 1982:183-96. 4. Preussmann R, Eisenbrand G. N-nitrosocarcinogens in the environment. In: Searle CE, ed. Chemical carcinogens Washington, DC: American Chemical Society, 1984:829-68 (ACS Monograph series 182.) S. Wagner DA, Shuker DEG, Bilmazes C, et al. Effect of vitamins C and F on endogenous synthesis of N-nitrosamino acids in humans: precursor-product studies with (lS-N)nitrate. Cancer Res l985;45: 65 19-22. 6. Hoffmann D, Brunnemann KD. Endogenous formation of N-nitrosoproline in cigarette smokers. Cancer Res l983;43:5570-74. 7. LeafCD, Vecchio Al, Hotchkiss IH. Influence ofascorbic acid dose on N-nitrosoproline formation in humans. Carcinogenesis l987;8: 79 1-5. 8. Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet Toxicol 1976; 14:545-8. 9. Perciballi M, Conboy II, Hotchkiss IH. Nitrite-cured meats as a source of endogenously and exogenously formed N-nitrosoproline in the ferret. Food Cosmet Toxicol 1989:27: 1 1 1-6.

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2495

INHIBITION

10. Neurath GB, Dunger M. Measurement ofnitrogen oxides in tobacco smoke by means of the chemiluminescence method. In: Bogovski P. Walker EA. eds. N-nitroso compounds in the environment. Lyon, France: International Agency for Research on Cancer. I 974:177-9 (IARC Scientific publication No 9.) I 1. Mirvish SS, Sams IP, Issenberg P. The nitrosating agent in mice exposed to nitrogen dioxide: improved extraction method and localization in the skin. Cancer Res l983;43:2550-4. 12. Garland WA, Kuenzig W, Rubio F, Kornychuk H, Norkus EP, Conney AH. Urinary excretion of nitrosodimethylamine and nitrosoproline in humans: interindividual and intraindividual differences and the effect of administered ascorbic acid and alpha-tocopherol. Cancer Res l986;46:5392-400. 13. Green LC, Wagner DA, Ruiz de Luzuriaga K, Istfan N, Young yR. Tannenbaum SR. Nitrate biosynthesis in man. Proc Natl Acad Sci USA 198 1;78:7764-8. 14. Green LC, Tannenbaum SR. Goldman P. Nitrate synthesis in the germfree and conventional rat. Science l98l;2l2:56-8. 15. Wagner DA, Young VR, Tannenbaum SR. Mammalian nitrate biosynthesis: incorporation of [1 5N]ammonia into nitrate is enhanced by endotoxin treatment. Proc NatI Acad Sci USA 1983:80:41821. 16. Wagner DA, Schultz DS, Deen WM, Young VR, Tannenbaum SR. Metabolic fate of an oral dose of ‘5N-labeled nitrate in humans: effect of diet supplementation with ascorbic acid. Cancer Res l983;43: 1921-S. 17. Wagner DA. Young VR, Tannenbaum SR. Schultz DS. Deen WM. Mammalian nitrate biochemistry: metabolism and endogenous synthesis. In: O’Neill 1K. von Borstel RC. eds. N-nitroso compounds: occurrence, biological effects and relevance to human cancer. Lyon, France: International Agency for Research on Cancer, 1984:24753 (IARC Scientific publication No 57.). 18. Stuehr DI, Marietta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia co/i lipopolysaccharide. Proc Natl Acad Sci USA l985;82:7738-42. 19. Stuehr DI, Marietta MA. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection lymphokines or interferongamma. I Immunol 1987:139:518-25. 20.

21

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Marletta MA, Yoon PS, Iyengar R, Leaf CD. Wishnok IS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry I 988;27:87061 1. Miwa M, Stuehr DI, Marietta MA, Wishnok IS, Tannenbaum SR. Nitrosation of amines by stimulated macrophages. Carcinogenesis l987;8:955-8.

22. Iyengar R, Stuehr DI, MarIetta MA. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst. Proc NatI Acad Sci USA l987;84:6369-73. 23.

Palmer RMJ, Ashton synthesize nitric oxide

DS, Moncada S. Vascular endothelial from L-arginine. Nature l988;333:664-6.

cells

24.

McCall TB, Boughton-Smith NK, Palmer RMJ, Whittle BJR, Moncada S. Synthesis of nitric oxide from L-arginine by neutrophils. Biochem I l989;261:293-6. 25. Knowles RG, Palacios M, Palmer RiM, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation ofthe soluble guanylate cyclase. Proc Natl Acad Sci USA l989;86:5159-62. 26.

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TANNENBAUM nitrite reduction by E coli Kl2. I Gen Microbiol l984;l30:127984. Ralt D. Tannenbaum SR. The role ofbacteria in nitrosamine formation. In: Scanlan RA, Tannenbaum SR, eds. N-nitroso compounds. Washington, DC: American Chemical Society, 1981:15764 (ACS Symposium series 174.). Suzuki 5, Mitsuoka T. N-nitrosamine formation by bacteria of the intestine. In: O’Neill 1K. von Borstel RC, LongJE, MillerCT, Bartsch H. eds. N-nitroso compounds: occurrence, biological effects, and relevance to human cancer. Lyon: International Agency for Research on Cancer, 1984:275-82 (IARC Scientific publication 57.). Leach SA, Cook AR, Challis BC, Hill MI, Thompson MH. Bacterially mediated N-nitrosation reactions and endogenous formation of N-nitroso compounds. In: Bartsch H, O’Neill I, Schulte-Hermann R. eds. The relevance of N-nitroso compounds to human cancer. Exposure and mechanisms. Lyon, France: International Agency for Research on Cancer, 1987:396-9 (IARC Scientific publication 84.). Calmels S. Ohshima H, Vincent P. Gounot AM, Bartsch H. Screening ofmicroorganisms for nitrosation catalysis at pH 7 and kinetic studies on nitrosamine formation from secondary amines by F. coli strains. Carcinogenesis 1985:6:91 1-5. RaIt D. Wishnok iS, Fitts R, Tannenbaum SR. Bacterial catalysis of nitrosation: involvement of the nar operon of Escherichia coli. I Bacteriol 1988: I 70:359-64. Calmels S. Ohshima H, Rosenkranz H, McCoy F, Bartsch H. Bio-

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ET AL

36. 37.

38.

39.

40.

4 1.

42.

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