Substrate

specificity

of

a

long-chain

alkylamine-degrading

Pseudomonas

sp

isolated

from

activated

sludge

A

bacterium

strain

BERT

which

utilizes

primary

long-chain

alkylamines

as

nitrogen

carbon

and

energy

source

was

isolated

from

activated

sludge

This

rod-shaped

motile

Gram-negative

strain

was

identified

as

a

Pseudomonas

sp.

The

substrate

spectrum

of

this

Pseudomonas

strain

BERT

includes

primary

alkylamines

with

alkyl

chains

ranging

from

to

and

Amines

with

alkyl

chains

ranging

from

to

carbons

were

the

preferred

substrates

Growth

on

dodecanal

dodecanoic

acid

and

acetic

acid

and

simultaneous

adaptation

studies

indicated

that

this

bacterium

initiates

degradation

through

a

Calkyl

N

cleavage

The

cleavage

of

alkylamines

to

the

respective

alkanals

in

Pseudomonas

strain

BERT

is

mediated

by

a

PMS-dependent

alkylamine

dehydrogenase

This

alkylamine

dehydrogenase

produces

stoichiometric

amounts

of

ammonium

from

octylamine

The

PMS-dependent

alkylamine

was

found

to

oxidize

a

broad

range

of

long-chain

alkylamines

PMS-dependent

long-chain

aldehyde

dehydrogenase

activity

was

also

detected

in

cell-free

extract

of

Pseudomonas

strain

BERT

grown

on

octylamine

The

proposed

pathway

for

the

oxidation

of

alkylamine

in

strain

BERT

proceeds

from

alkylamine

to

alkanal

and

then

to

the

fatty

acid

Introduction

Primary

fatty

amines

contain

a

nitrogen

atom

attached

to

one

long

alkyl

chain

Commercial

primary

alkylamines

are

usually

mixtures

of

homologs

because

the

sources

of

the

hydrophobic

groups

are

fatty

acids

derived

from

palm

oil

coconut

oil

or

tallow

The

alkyl

chains

therefore

vary

in

both

chain

length

and

degree

of

unsaturation

Alkylamines

are

primarily

introduced

into

the

environment

through

emissions

during

industrial

production

The

alkylamines

produced

in

high

volumes

are

toxic

to

aquatic

organisms

-LRB-

Newsome

et

al.

Schultz

et

al.

Finlay

and

Callow

-RRB-

Microbial

degradation

of

primary

fatty

amines

is

therefore

important

for

removal

in

biological

wastewater

treatment

systems

and

to

maintain

low

environmental

concentrations

Yoshimura

et

al.

-LRB-

-RRB-

studied

the

biodegradability

of

various

fatty

amines

in

the

MITI

test

The

MITI

test

assesses

the

ready

biodegradability

by

determining

the

biological

oxygen

demand

-LRB-

OECD

-RRB-

Ratios

of

the

biological

oxygen

demand

and

theoretical

oxygen

demand

were

achieved

in

MITI

tests

demonstrating

the

susceptibility

of

alkylamines

-LRB-

to

-RRB-

to

biodegradation

-LRB-

Yoshimura

et

al.

-RRB-

Dodecylamine

was

also

demonstrated

to

be

biodegradable

in

another

ready

biodegradability

test

i.e.

Closed

Bottle

test

-LRB-

OECD

-RRB-

The

biodegradation

of

dodecylamine

started

immediately

and

reached

within

a

week

-LRB-

van

Ginkel

et

al.

-RRB-

The

results

obtained

in

ready

biodegradability

tests

are

far

more

satisfactory

when

supported

by

pure

culture

studies

Until

now

two

pure

cultures

of

bacteria

have

been

shown

to

degrade

primary

fatty

amines

-LRB-

Yoshimura

et

al.

Selig

et

al.

-RRB-

Possible

intermediates

of

alkylamine

degradation

were

not

identified

in

these

studies

Therefore

much

remains

to

be

learned

about

the

substrate

specificity

of

long-chain

alkylamine

degrading

micro-organisms

and

the

biodegradation

pathway

The

aim

of

this

study

was

to

investigate

how

alkylamines

are

degraded

by

micro-organisms

using

a

pure

culture

capable

of

utilizing

alkylamines

as

sole

carbon

and

energy

source

Substrate

specificities

of

the

pure

culture

and

cell-free

extracts

catalysing

the

initial

degradation

steps

are

reported

Materials

and

methods

Chemicals

Samples

of

octylamine

decylamine

dodecylamine

tetradecylamine

hexadecylamine

octadecylamine

cocoamine

and

tallowamine

-LRB-

Armeen

-RRB-

were

provided

by

Akzo

Nobel

Surfactants

Stenungsund

Sweden

All

other

chemicals

were

of

reagent-grade

quality

and

obtained

from

Sigma

Aldrich

Zwijndrecht

The

Netherlands

or

Akcross

Organics

Geel

Belgium

The

biochemicals

were

purchased

from

Boehringer

Mannheim

Germany

Activated

sludge

Activated

sludge

used

as

inoculum

was

obtained

from

the

wastewater

treatment

plant

Nieuwgraaf

in

Duiven

The

Netherlands

This

activated

sludge

plant

treats

predominantly

domestic

wastewater

Media

The

mineral

salts

medium

used

for

isolation

and

growth

experiments

contained

the

following

in

l

of

deionized

water

g

g

g

g

and

ml

trace

solution

described

by

Vishniac

and

Santer

-LRB-

-RRB-

The

medium

was

sterilized

by

autoclaving

for

min

together

with

any

added

growth

substrate

-LRB-

g

l

-RRB-

The

volatile

primary

amines

i.e.

octylamine

and

decylamine

were

added

after

autoclaving

the

mineral

salt

medium

When

these

volatile

primary

amines

were

used

as

growth

substrate

silica

gel

was

added

to

give

a

final

concentration

of

g

l

Silica

gel

was

added

to

reduce

the

concentration

of

alkylamines

in

the

water

phase

Enrichment

isolation

and

growth

Aerobic

long-chain

alkylamine

degrading

micro-organisms

were

isolated

from

an

enrichment

culture

developed

from

activated

sludge

The

enrichment

culture

was

obtained

in

batch

culture

flasks

containing

ml

mineral

salts

medium

and

g

l

dodecylamine

inoculated

with

ml

of

activated

sludge

After

five

subcultures

a

bacterium

in

this

culture

was

streaked

to

purity

on

agar

plates

containing

mineral

salts

medium

g

l

dodecylamine

and

g

l

agar

The

cultures

and

agar

plates

were

incubated

at

°C

The

growth

rate

of

strain

BERT

was

recorded

by

measuring

the

increase

in

turbidity

in

culture

flasks

with

a

Hach

Ratio

XR

turbidimeter

-LRB-

Hach

Loveland

CO

USA

-RRB-

The

ability

of

the

strain

to

grow

on

other

substrates

was

tested

using

similar

media

in

which

dodecylamine

was

substituted

with

possible

intermediates

or

other

fatty

amine

derivatives

Culture

conditions

and

preparation

of

washed

cell

suspensions

and

cell-free

extracts

Cells

were

grown

on

octylamine

as

sole

nitrogen

carbon

and

energy

source

in

a

continuous

culture

This

continuous

culture

was

run

at

°C

in

a

fermentor

-LRB-

Applikon

Schiedam

The

Netherlands

-RRB-

with

a

working

volume

of

l

The

impeller

speed

was

rpm

The

mineral

salt

medium

was

pumped

continuously

into

the

fermentor

at

a

rate

of

l

day

by

means

of

a

peristaltic

pump

Octylamine

was

introduced

separately

to

the

fermentor

with

a

syringe

pump

and

teflon

tube

giving

a

final

concentration

of

g

l

This

set-up

was

required

because

of

transport

of

the

alkylamine

through

silicone

tubes

Cells

were

also

cultivated

in

the

fermentor

with

acetate

octanal

and

octanoate

under

the

same

conditions

but

with

a

mineral

salts

medium

containing

the

respective

organic

compounds

The

pH

of

the

reactor

was

maintained

at

Experiments

on

the

nitrogen

balance

were

also

conducted

in

the

fermentor

fed

with

a

mineral

salts

medium

without

ammonium

chloride

The

nitrogen

recovery

was

estimated

from

the

amount

of

nitrogen

leaving

the

fermentor

as

biomass

ammonium

and

total

nitrogen

The

total

nitrogen

represents

the

residual

substrate

and/or

water-soluble

nitrogen-containing

compound

formed

during

the

biodegradation

process

Cells

were

harvested

from

the

continuous

cultures

by

centrifugation

at

g

for

min

and

washed

three

times

with

mM

phosphate

buffer

pH

Washed

cells

-LRB-

ml

-RRB-

were

disrupted

by

a

French

pressure

cell

at

psi

-LRB-

Thermo

IEC

Needham

Heights

MA

USA

-RRB-

After

one

pass

the

insoluble

matter

was

precipitated

at

g

for

min

Oxygen

consumption

The

endogenous

and

substrate-dependent

oxygen

uptake

rates

by

washed

cell

suspensions

were

determined

in

a

polarographic

oxygen

monitor

-LRB-

Yellow

Springs

Instruments

Yellow

Springs

OH

USA

-RRB-

The

polarographic

oxygen

monitor

consisted

of

a

thermostated

vessel

with

a

magnetic

stirrer

and

an

oxygen

electrode

to

measure

oxygen

depletion

Cells

were

resuspended

in

phosphate

buffer

at

°C

in

a

final

volume

of

ml

After

min

necessary

to

determine

the

endogenous

respiration

the

substrate-dependent

respiration

was

measured

by

injection

of

ml

of

a

g

l

substrate

solution

into

the

vessel

Suspensions

of

alkylamines

with

more

than

ten

carbons

and

octanal

were

heated

prior

to

injection

Enzyme

assays

All

enzyme

assays

were

performed

at

°C

Spectrophotometric

enzyme

assays

were

carried

out

in

a

Shimadzu

UV

spectrophotometer

-LRB-

Shimadzu

Kyoto

Japan

-RRB-

in

cm

light-path

cuvettes

The

oxygen

uptake

was

measured

with

a

polarographic

oxygen

monitor

-LRB-

Yellow

Springs

Instruments

-RRB-

Alkylamine

dehydrogenase

-LRB-

phenazinium

methyl

sulfate

-LRB-

PMS

-RRB-

dependent

-RRB-

was

assayed

by

recording

the

oxygen

uptake

in

a

polarographic

oxygen

monitor

The

reaction

mixture

-LRB-

volume

ml

-RRB-

contained

mM

PMS

and

g

l

protein

in

a

phosphate

buffer

-LRB-

mM

pH

-RRB-

The

reaction

was

started

by

injecting

alkylamine

in

the

reaction

vessel

at

a

concentration

of

mM

Alkylamine

dehydrogenase

-LRB-

PMS-dependent

-RRB-

activity

was

also

determined

by

colorimetric

detection

of

ammonium

in

a

reaction

vessel

containing

mM

octylamine

mM

PMS

and

g

l

protein

in

a

phosphate

buffer

-LRB-

mM

pH

-RRB-

The

enzyme

assays

were

carried

out

at

least

twice

and

the

difference

in

specific

activity

was

less

than

Alkylamine

dehydrogenase

-LRB-

NAD

-LRB-

P

-RRB-

dependent

-RRB-

activity

was

spectrophotometrically

assayed

by

measuring

the

increase

of

NAD

-LRB-

P

-RRB-

H

at

nm

The

reaction

mixture

consisted

of

mM

NAD

-LRB-

P

-RRB-

a

phosphate

buffer

-LRB-

mM

pH

-RRB-

and

cell-free

extract

-LRB-

g

l

protein

-RRB-

in

a

total

volume

of

ml

The

alkylamine

dehydrogenase

-LRB-

NAD

-LRB-

P

-RRB-

dependent

-RRB-

activity

was

measured

under

anaerobic

conditions

to

minimize

NADH

oxidase

activity

For

that

purpose

the

contents

of

the

cuvettes

were

stoppered

with

Suba

Seal

septa

-LRB-

Sigma

Aldrich

Zwijndrecht

The

Netherlands

-RRB-

and

flushed

with

nitrogen

gas

Aldehyde

dehydrogenase

activity

was

also

measured

by

spectrophotometric

and

a

respirometric

assays

Aldehyde

dehydrogenase

-LRB-

PMS-dependent

-RRB-

activity

was

assayed

respirometrically

The

reaction

mixture

-LRB-

volume

ml

-RRB-

contained

a

phosphate

buffer

-LRB-

mM

pH

-RRB-

mM

PMS

cell-free

extract

-LRB-

g

l

protein

-RRB-

The

reaction

was

started

by

injecting

octanal

in

the

reaction

vessel

giving

mM

Aldehyde

dehydrogenase

-LRB-

NAD

-LRB-

P

-RRB-

dependent

-RRB-

activity

was

determined

by

measuring

the

increase

in

absorbance

at

nm

Reaction

mixtures

contained

phosphate

buffer

-LRB-

mM

pH

-RRB-

μM

aldehyde

mM

NAD

and

mM

KCN

and

enzyme

solution

in

a

volume

of

ml

Analyses

Ammonium

was

determined

colorimetrically

by

forming

indophenol

blue

with

hypochlorite

and

salicylate

in

the

presence

of

sodium

nitroferricyanide

as

catalyst

-LRB-

Verdouw

et

al.

-RRB-

Protein

was

quantified

by

bicinchoninic

acid

method

Cells

were

first

lysed

by

incubating

at

°C

with

M

NaOH

The

protein

concentration

was

estimated

by

using

the

Bio-Rad

Protein

assay

kit

with

bovine

serum

albumin

as

standard

protein

Dissolved

organic

carbon

and

total

nitrogen

were

quantified

with

a

Shimadzu

TOC

apparatus

-LRB-

Shimadzu

-RRB-

Octylamine

was

analysed

by

ion

chromatography

with

a

Dionex

-LRB-

Dionex

Sunnyvale

CA

USA

-RRB-

The

Dionex

was

equipped

with

an

IonPac

-LRB-

mm

-RRB-

analytical

column

a

μl

sample

loop

a

CSRS-I

in

the

external

water

mode

and

a

flow-through

conductivity

cell

with

a

detection

stabilizer

The

was

operated

at

a

column

temperature

of

°C

and

a

detector

temperature

of

°C

The

eluent

was

deionized

water

with

mM

methylsulphonic

acid

and

acetonitrile

The

eluent

flow

rate

was

ml

min

The

dry

weight

of

the

micro-organisms

was

determined

by

washing

and

concentrating

a

known

volume

from

the

continuous

culture

through

centrifugation

-LRB-

g

for

min

-RRB-

The

concentrated

biomass

on

a

preweighed

watch

glass

was

dried

for

h

at

°C

Results

Isolation

and

characterization

A

culture

capable

of

growth

on

dodecylamine

was

readily

enriched

from

activated

sludge

through

repeated

transfers

of

cells

to

fresh

mineral

medium

containing

dodecylamine

Dodecylamine

plates

streaked

with

dilutions

of

this

enrichment

culture

enabled

the

isolation

of

bacteria

The

predominant

isolate

BERT

was

a

Gram-negative

motile

non-spore-forming

rod

μm

long

and

μm

wide

Oxidase

catalase

urease

and

alcohol

dehydrogenase

were

present

The

strain

was

able

to

utilize

the

following

substrates

glucose

phenylacetate

citrate

malate

mannose

mannitole

and

gluconate

It

did

not

grow

on

maltose

trehalose

m-inositol

citraconate

erytritol

sorbitol

d-xylose

d-tartrate

and

l-arabinose

The

strain

was

not

capable

of

reducing

nitrate

The

identity

of

the

primary

alkylamine

degrading

bacterium

was

determined

by

the

profile

of

cellular

fatty

acids

and

partial

rRNA

gene

sequencing

The

profile

of

the

cellular

fatty

acids

is

typical

for

the

RNA

group

I

of

the

genus

Pseudomonas

The

partial

sequences

have

shown

a

similarity

of

to

Pseudomonas

putida

P.

plecoglosscida

and

P.

alcaligenes

These

characteristics

only

allow

to

place

strain

BERT

within

the

RNA

group

I

of

the

genus

Pseudomonas

Growth

A

number

of

fatty

amine

derivatives

were

tested

for

their

ability

to

support

growth

of

Pseudomonas

strain

BERT

The

strain

was

found

to

be

able

to

grow

on

a

wide

range

of

long-chain

primary

amines

i.e.

octylamine

-LRB-

in

the

presence

of

silica

gel

-RRB-

decylamine

-LRB-

in

the

presence

of

silica

gel

-RRB-

dodecylamine

tetradecylamine

octadecylamine

cocoamine

oleylamine

and

tallowamine

as

sole

source

of

carbon

and

both

in

the

presence

of

silica

gel

were

also

used

as

carbon

and

energy

source

by

Pseudomonas

strain

BERT

whereas

dodecyldimethylamine

didodecylamine

didodecymethylamine

dodecyltrimethylammonium

chloride

did

not

support

growth

Mineral

salts

media

amended

with

possible

intermediates

of

dodecylamine

degradation

such

as

dodecanal

dodecanoic

acid

and

acetate

supported

growth

of

Pseudomonas

strain

BERT

Depletion

of

dodecylamine

to

non-detectable

concentrations

from

the

nitrogen-free

mineral

salts

medium

in

batch

cultures

and

the

concurrent

growth

of

the

isolate

clearly

demonstrate

that

this

isolate

also

used

alkylamines

as

its

nitrogen

source

-LRB-

data

not

shown

-RRB-

Octylamine

was

used

as

growth

substrate

in

a

number

of

experiments

because

this

amine

is

soluble

in

water

at

a

concentration

of

g

l

Use

of

octylamine

allowed

for

instance

accurate

measurement

of

the

increase

in

turbidity

due

to

growth

From

the

growth

curve

a

doubling

time

of

h

was

estimated

-LRB-

data

not

shown

-RRB-

Growth

yield

obtained

with

Pseudomonas

strain

BERT

was

g

dry

weight

of

cells

per

g

of

octylamine

utilized

The

fate

of

the

nitrogen

of

octylamine

in

terms

of

ammonium

biomass-nitrogen

nitrogen-containing

soluble

microbial

products

and

residual

octylamine

was

assessed

in

a

continuous

culture

Total

nitrogen

ammonium

and

biomass-nitrogen

were

measured

in

triplicate

Approximately

of

the

nitrogen

was

recovered

as

biomass-nitrogen

Octylamine-nitrogen

was

converted

for

into

ammonium

Less

than

of

the

nitrogen

in

the

effluent

was

present

as

octylamine

Approximately

of

the

nitrogen

was

recovered

as

water-soluble

nitrogen-containing

products

Respiration

experiments

The

alkylamine-degrading

isolate

was

grown

on

a

number

of

different

substrates

as

sole

source

of

carbon

after

which

respiration

rates

of

washed

cell

suspensions

were

examined

with

a

variety

of

substrates

in

order

to

identify

possible

intermediates

of

the

degradation

pathway

-LRB-

Table

-RRB-

Washed

cell

suspensions

of

Pseudomonas

strain

BERT

grown

with

octylamine

were

capable

of

oxidizing

octylamine

octanoate

octanal

and

acetate

Whole

cells

of

Pseudomonas

strain

BERT

also

showed

octylamine-dependent

oxygen

uptake

when

grown

with

octanal

and

octanoate

However

strain

BERT

grown

with

acetate

did

not

respire

octylamine

Finally

Pseudomonas

strain

BERT

grown

on

octanal

octanoate

and

actate

displayed

significant

activities

with

the

alkanal

and

alkanoate

tested

-LRB-

Table

-RRB-

Table

of

various

potential

intermediates

of

alkylamine

degradation

by

washed

cell

suspensions

of

strain

BERT

grown

on

octylamine

octanal

octanoate

and

acetateSubstrateGrowth

substrateOctylamineOctanalOctanoateAcetatenmol

min

mg

of

oxygen

uptake

are

expressed

as

nmoles

min

mg

protein

after

correction

for

endogenous

respiration

The

endogenous

respiration

of

the

strain

grown

on

octylamine

octanal

octanoate

and

acetate

were

and

nmol

min

mg

protein

respectively

The

ability

of

octylamine-grown

strain

BERT

to

oxidize

alkylamines

was

also

evaluated

-LRB-

Table

-RRB-

Octylamine-grown

cells

were

capable

of

oxidizing

all

alkylamines

with

alkyl

chains

ranging

from

to

carbon

atoms

The

highest

activity

was

observed

when

hexylamine

octylamine

and

decylamine

were

added

to

the

reaction

vessel

The

oxidation

rate

of

nonylamine

was

comparable

to

the

activity

found

with

octylamine

Propylamine

was

respired

at

a

low

rate

Low

activities

were

also

detected

with

alkylamines

with

more

than

carbon

atoms

All

other

fatty

amine

derivatives

except

for

were

not

oxidized

by

strain

BERT

grown

on

octylamine

-LRB-

Table

-RRB-

Table

of

various

alkylamines

by

washed

cell

suspensions

and

alkylamine

dehydrogenase

-LRB-

PMS-dependent

-RRB-

SubstrateWashed

cell

suspensionCell-free

extractnmol

min

mg

of

oxygen

uptake

are

expressed

as

nmoles

min

mg

protein

The

rates

for

the

washed

cell

suspensions

are

corrected

for

the

endogenous

respiration

of

octylamine-grown

cells

i.e.

nmol

min

mg

protein

Enzymatic

activities

Activities

of

enzymes

that

may

be

involved

in

alkylamine

metabolism

were

tested

in

cell-free

extracts

prepared

from

cells

grown

on

octylamine

Extracts

of

octylamine

grown

cells

released

ammonium

in

stoichiometric

amounts

from

octylamine

in

the

presence

of

PMS

-LRB-

Fig.

-RRB-

In

another

experiment

the

presence

of

PMS

induced

the

consumption

of

mM

oxygen

upon

the

addition

of

mM

octylamine

The

activity

of

the

PMS-dependent

alkylamine

dehydrogenase

with

dodecylamine

was

nmol

min

mg

protein

NAD

and

NADP

could

not

replace

the

artificial

electron

acceptor

The

substrate

specificity

of

cell-free

extracts

catalysing

the

conversion

of

alkylamines

was

studied

by

measuring

the

oxygen

consumption

in

the

presence

of

PMS

The

enzyme

was

active

towards

alkylamines

with

alkyl

chains

with

carbons

Propylamine

nonylamine

and

also

acted

as

substrate

for

the

enzyme

Among

the

primary

alkylamines

examined

the

most

preferred

were

alkylamines

with

alkyl

chains

length

ranging

from

to

carbons

Methylamine

secondary

tertiary

and

quaternary

fatty

amine

derivatives

were

no

adequate

substrates

-LRB-

Table

-RRB-

Demonstration

of

alkanal

dehydrogenase

activity

in

cell

extracts

was

achieved

by

incubating

cell-free

extracts

with

octanal

and

PMS

and

measuring

the

oxygen

consumption

An

activity

of

nmol

min

mg

protein

was

measured

Alkanal

dehydrogenase

activity

was

not

detected

with

other

electron

acceptors

i.e.

NAD

and

NADP

despite

repeated

attempts

Fig.

of

ammonium

from

mM

octylamine

by

cell-free

extract

of

octylamine-grown

cells

in

the

presence

-LRB-

open

square

-RRB-

and

absence

-LRB-

filled

square

-RRB-

of

PMS

The

protein

concentration

in

the

assay

was

g

l

Discussion

The

fate

of

long-chain

alkylamines

in

the

environment

is

largely

dependent

on

the

ability

of

micro-organisms

to

metabolize

these

compounds

Although

degradation

of

alkylamines

in

OECD

ready

biodegradability

tests

has

been

observed

-LRB-

Yoshimura

et

al.

OECD

van

Ginkel

et

al.

-RRB-

little

is

known

about

the

intermediates

in

the

biodegradation

process

and

the

enzymes

involved

in

the

biodegradation

In

this

paper

we

describe

the

properties

of

the

newly

isolated

Pseudomonas

strain

BERT

which

is

capable

of

degrading

long-chain

alkylamines

Yoshimura

et

al.

-LRB-

-RRB-

also

isolated

a

Pseudomonas

strain

Both

Pseudomonas

strains

degraded

primary

alkylamines

with

varying

alkyl

chain

lengths

Pseudomonas

species

are

noted

for

their

metabolic

diversity

and

are

often

isolated

from

enrichments

designed

to

identify

bacteria

that

degrade

anthropogenic

organic

compounds

Another

species

known

to

utilize

a

long-chain

alkylamine

as

sole

source

of

carbon

and

energy

is

from

the

genus

Rhodococcus

isolated

from

top

soil

-LRB-

Selig

et

al.

-RRB-

A

nitrogen

mass

balance

obtained

in

a

continuous

culture

already

strongly

indicated

that

octylamine

a

water

soluble

long-chain

alkylamine

is

completely

-LRB-

ultimately

-RRB-

biodegradable

because

almost

all

of

the

octylamine-nitrogen

was

converted

into

biomass-nitrogen

and

ammonium

Other

evidence

of

complete

degradation

of

alkylamines

has

been

obtained

through

the

proposed

biodegradation

pathway

The

biodegradation

of

alkylamines

was

assumed

to

proceed

either

via

a

Calkyl

N

cleavage

or

oxidation

at

the

far-end

of

the

alkyl

chain

Based

on

the

induction

pattern

and

PMS-dependent

dehydrogenase

activity

with

alkylamines

the

biodegradation

of

alkylamines

is

suspected

to

involve

a

cleavage

of

the

Calkyl

N

bond

which

yield

the

respective

alkanals

and

ammonium

The

alkanals

are

oxidized

by

a

PMS-dependent

alkanal

dehydrogenase

to

the

respective

fatty

acids

The

induction

of

long-chain

alkanal

dehydrogenase

activity

by

growth

of

the

bacterium

on

octylamine

demonstrates

that

alkanals

are

significant

intermediates

A

PMS-dependent

long-chain

alkanal

dehydrogenase

was

also

detected

in

Acinetobacter

calcoaceticus

grown

on

hexadecane

and

hexadecanol

-LRB-

Fox

et

al.

-RRB-

At

present

it

is

unknown

if

both

the

alkanal

and

the

alkylamine

are

dehydrogenated

by

the

same

enzyme

in

Pseudomonas

strain

BERT

Fatty

acids

produced

by

the

alkanal

dehydrogenase

are

channelled

into

the

β-oxidation

cycle

-LRB-

Ratledge

-RRB-

The

degradative

pathway

shown

in

Fig.

is

inducible

because

octylamine

is

not

oxidized

by

acetate-grown

cells

-LRB-

Table

-RRB-

The

degradation

pathway

is

quite

similar

to

those

described

for

alkyltrimethylammonium

salts

alkyldimethylamines

and

alkylbis

-LRB-

-RRB-

amines

-LRB-

van

Ginkel

-RRB-

This

biodegradation

pathway

provides

evidence

of

total

mineralization

of

alkylamines

as

indicated

by

the

nitrogen

mass

balance

and

the

ready

biodegradability

test

results

-LRB-

Yoshimura

et

al.

van

Ginkel

et

al.

-RRB-

Fig.

pathway

for

the

degradation

of

alkylamines

in

Pseudomonas

strain

BERT

-LRB-

-RRB-

alkylamine

dehydrogenase

-LRB-

-RRB-

aldehyde

dehydrogenase

The

fatty

acids

formed

are

channelled

into

the

β-oxidation

cycle

PMS

an

artificial

electron

acceptor

is

generally

used

to

assay

enzymes

known

as

quinoprotein

enzymes

Physiological

electron

acceptors

are

cytochrome

c

protein-bound

haem

and

ubiquinone

Methylamine

dehydrogenases

-LRB-

de

Beer

et

al.

Duine

et

al.

-RRB-

short-chain

alkylamine

dehydrogenases

-LRB-

Shinagawa

et

al.

-RRB-

and

a

long-chain

alkanal

dehydrogenase

-LRB-

Fox

et

al.

-RRB-

have

been

identified

as

quinoproteins

Methylamine

dehydrogenases

catalyse

the

oxidative

deamination

of

methylamine

to

methanal

and

ammonium

-LRB-

McIntire

et

al.

-RRB-

The

PMS

dependent

removal

of

ammonium

from

alkylamines

in

strain

BERT

may

be

catalysed

by

an

enzyme

similar

to

methylamine

dehydrogenases

although

no

activity

towards

methylamine

was

detected

-LRB-

Table

-RRB-

Zhu

et

al.

-LRB-

-RRB-

demonstrated

that

the

conversion

of

one

amino

acid

of

methylamine

dehydrogenase

altered

the

substrate

preference

of

methylamine

dehydrogenase

to

a

large

extent

The

preferred

substrates

of

the

mutated

enzyme

are

alkylamines

with

at

least

seven

carbons

Long-chain

alkylamines

are

probably

also

oxidized

by

an

alkylamine

dehydrogenase

isolated

from

a

butylamine-grown

Pseudomonas

sp.

although

the

highest

activity

was

found

with

butylamine

-LRB-

Shinagawa

et

al.

-RRB-

A

quinoprotein

was

also

found

in

a

P.

putida

catalysing

the

deamination

of

propylamine

and

butylamine

both

naturally

occurring

alkylamines

-LRB-

Adachi

et

al.

-RRB-

Only

a

long-chain

alkylamine

dehydrogenase

is

required

to

convert

anthropogenic

alkylamines

into

compounds

which

can

enter

an

existing

pathway

This

enzyme

may

have

evolved

from

methylamine

dehydrogenases

or

short-chain

alkylamine

dehydrogenases

involved

in

the

degradation

of

naturally

occurring

amines

A

major

feature

of

Pseudomonas

strain

BERT

is

its

broad

substrate

specificity

with

respect

to

primary

alkylamines

Activities

of

washed

cell

suspensions

and

alkylamine

dehydrogenase

activities

in

cell-free

extracts

demonstrate

that

alkylamines

with

alkyl

chain

lengths

ranging

from

to

carbons

are

preferred

Odd

alkyl

chain

amines

were

also

respired

by

Pseudomonas

strain

BERT

Water-insoluble

long-chain

alkylamines

are

oxidized

at

a

lower

rate

by

washed

cell

suspensions

and

alkylamine

dehydrogenase

of

strain

BERT

This

probably

reflects

the

low

bioavailability

of

the

long-chain

alkylamines

Limited

bioavailability

of

long-chain

alkylamines

-LRB-

to

-RRB-

can

also

be

concluded

from

the

non-toxicity

of

the

alkylamines

to

strain

BERT

at

a

concentration

of

g

l

In

batch

cultures

growth

of

Pseudomonas

strain

BERT

on

octylamine

and

decylamine

was

only

possible

in

the

presence

of

silica

gel

Silica

gel

reduces

the

toxicity

of

fatty

amine

derivatives

through

adsorption

onto

the

silica

gel

-LRB-

van

Ginkel

et

al.

Selig

et

al.

-RRB-

The

ease

with

which

Pseudomonas

strain

BERT

was

isolated

and

the

high

growth

rate

of

strain

BERT

are

consistent

with

studies

showing

ready

biodegradability

of

many

primary

amines

-LRB-

Yoshimura

et

al.

van

Ginkel

et

al.

-RRB-

Based

on

the

biodegradation

pathway

and

the

broad

substrate

specificity

significant

differences

in

the

biodegradability

of

alkylamines

with

varying

chain

lengths

are

not

expected

However

degradation

of

primary

alkylamines

does

differ

in

ready

biodegradability

tests

-LRB-

Yoshimura

et

al.

-RRB-

This

can

be

explained

by

biocidal

effects

and

limited

bioavailability

Reading

across

of

the

ready

biodegradability

test

results

enabled

by

this

study

reveals

that

all

long-chain

alkylamines

both

saturated

and

unsaturated

are

readily

biodegradable

Future

research

should

focus

on

identifying

the

intermediates

and

purifying

the

enzymes

involved

in

the

degradation

of

alkylamines

B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

pseudomonas sp
substrate specificity
biodegradation pathway


Characteristics of Sexual Abuse in Childhood and Adolescence Influence Sexual Risk Behavior in Adulthood Childhood and adolescent sexual abuse has been associated with subsequent -LRB- adult -RRB- sexual risk behavior , but the effects of force and type of sexual abuse on sexual behavior outcomes have been less well-studied . The present study investigated the associations between sexual abuse characteristics and later sexual risk behavior , and explored whether gender of the child/adolescent moderated these relations . Patients attending an STD clinic completed a computerized survey that assessed history of sexual abuse as well as lifetime and current sexual behavior . Participants were considered sexually abused if they reported a sexual experience -LRB- 1 -RRB- before age 13 with someone 5 or more years older , -LRB- 2 -RRB- between the ages of 13 and 16 with someone 10 or more years older , or -LRB- 3 -RRB- before the age of 17 involving force or coercion . Participants who were sexually abused were further categorized based on two abuse characteristics , namely , use of penetration and force . Analyses included 1177 participants -LRB- n = 534 women ; n = 643 men -RRB- . Those who reported sexual abuse involving penetration and/or force reported more adult sexual risk behavior , including the number of lifetime partners and number of previous STD diagnoses , than those who were not sexually abused and those who were abused without force or penetration . There were no significant differences in sexual risk behavior between nonabused participants and those who reported sexual abuse without force and without penetration . Gender of the child/adolescent moderated the association between sexual abuse characteristics and adult sexual risk behavior ; for men , sexual abuse with force and penetration was associated with the greatest number of episodes of sex trading , whereas for women , those who were abused with penetration , regardless of whether the abuse involved force , reported the most episodes of sex trading . These findings indicate that more severe sexual abuse is associated with riskier adult sexual behavior . Introduction Childhood and adolescent sexual abuse has been associated with a wide variety of adverse mental and physical health outcomes . Research also suggests that the greater the severity of the sexual abuse , the worse the health outcomes . Thus , more severe sexual abuse -LRB- e.g. , sexual abuse involving force , more intimate sexual acts , a close relative , or repeated sexual abuse -RRB- has been associated with poorer social adjustment , less life satisfaction , and more severe psychological symptoms -LRB- Callahan , Price , & Hilsenroth , 2003 ; Carlson , McNutt , & Choi , 2003 ; Fassler , Amodeo , Griffin , Clay , & Ellis , 2005 ; Feinauer , Mitchell , Harper , & Dane , 1996 -RRB- . In a meta-analysis on the effects of child sexual abuse , Rind , Tromovitch , and Bauserman -LRB- 1998 -RRB- found that force was associated with more negative reactions but not with later psychological symptoms , whereas penetration was unrelated to these outcomes . Sexual abuse severity also has been associated with subsequent sexual risk behavior , including more sexual partners -LRB- Merrill , Guimond , Thomsen , & Milner , 2003 -RRB- and greater likelihood of sex with someone just met , earlier age at first intercourse , and a higher frequency of STD diagnoses -LRB- Walser & Kern , 1996 -RRB- . Although these studies suggest that more severe sexual abuse is associated with more sexual risk behavior , they provide only limited information regarding whether specific aspects of the abuse predict such outcomes . Needed is more fine-grained research to determine whether characteristics of the abuse experience -LRB- e.g. , whether physical force was used during the abuse and the type of sexual act that occurred during the abuse -RRB- are associated with sexual health outcomes . Two studies have examined the association between sexual abuse with force and later sexual risk behavior . Cinq-Mars , Wright , Cyr , and McDuff -LRB- 2003 -RRB- found that adolescent girls who experienced childhood or adolescent sexual abuse with force were more likely than girls who were sexually abused without force to have engaged in subsequent consensual sex during adolescence , to have more than one partner per year , and to have been pregnant . In a sample of men who have sex with men -LRB- MSM -RRB- , Jinich et al. -LRB- 1998 -RRB- reported that sexual abuse perceived as moderately or strongly coerced was associated with higher frequency of unprotected anal sex and higher HIV seroprevalence rates , relative to MSM who perceived the sexual abuse as voluntary or mildly coerced . Thus , in these two studies , sexual abuse involving force has been associated with greater sexual risk behavior . Investigations of the effects of penetrative -LRB- vs. non-penetrative -RRB- sexual abuse have yielded mixed results . Cinq-Mars et al. -LRB- 2003 -RRB- found that adolescent girls who experienced sexual abuse involving penetration were more likely to have engaged in consensual sex and experienced an unplanned pregnancy , compared to girls who experienced non-penetrative sexual abuse . Fergusson , Horwood , and Lynskey -LRB- 1997 -RRB- found that , compared to participants reporting no sexual abuse , those who experienced non-penetrative sexual abuse reported higher rates of unprotected sex . However , those who experienced penetrative sexual abuse reported the worst outcomes ; compared to participants who were not sexually abused , those who reported penetrative sexual abuse were more likely to have been pregnant , to report more than five sexual partners by age 18 , to have had unprotected sex , and to have had an STD . In contrast , in a meta-analysis , Arriola , Louden , Doldren , and Fortenberry -LRB- 2005 -RRB- found that the effect size for the relation between sexual abuse and later sexual behavior -LRB- i.e. , unprotected sex , sex with multiple partners , and sex work -RRB- did not differ for studies including non-contact abuse , studies including contact abuse only , and studies including penetration abuse only ; their findings suggest that type of sexual act during sexual abuse was not associated with later sexual behavior . However , some of these categories included very few studies -LRB- e.g. , there were only three studies that included penetration abuse only -RRB- . Furthermore , effects may have been obscured if studies with less restrictive definitions of sexual abuse included a large number of participants who had exper - ienced more severe -LRB- i.e. , contact or penetrative -RRB- sexual abuse . In sum , evidence from a small number of studies suggests that force and penetration may be associated with adult sexual risk behavior . An important limitation of previous research is that few studies have investigated the differential effects of sexual abuse for men and women . Because women in heterosexual relationships often have less control or power over sexual encounters compared to men -LRB- see the Theory of Gender and Power , Connell , 1987 , for an explanation of the power imbalance between men and women -RRB- , it is important to study gender in relation to sexual health behaviors . Indeed , the limited research on this topic suggests that the association between sexual abuse and adult sexual behavior differs by gender -LRB- e.g. , Futterman , Hein , Reuben , Dell , & Shaffer , 1993 ; Mason , Zimmerman , & Evans , 1998 ; Zierler et al. , 1991 -RRB- . It is possible that gender interacts with abuse characteristics to lead to different outcomes for males and females , an idea which is supported by research on the psychological sequelae of sexual abuse . For example , in a meta-analysis , Rind et al. -LRB- 1998 -RRB- found that whether or not the sexual abuse experience was consensual was associated with later psychological adjustment for men , but not for women . Few studies have investigated the effects of the interaction of gender and abuse characteristic on later sexual behavior . Overall , previous studies investigating the association between abuse characteristics and later sexual behavior have tended to use small samples , or included only males or only females , thus precluding gender comparisons . The primary purpose of this study was to determine whether use of force and type of sexual act was associated with sexual risk behavior in a group of patients receiving outpatient care from a sexually transmitted disease -LRB- STD -RRB- clinic . Based on previous research , we hypothesized that : -LRB- 1 -RRB- the use of force ; and -LRB- 2 -RRB- sexual abuse involving penetration would be associated with greater sexual risk behavior . The secondary purpose of this study was to determine whether the effects of abuse characteristics on adult sexual behavior differed by gender . Method Participants Participants were men and women attending a public STD clinic in upstate New York . All had been screened for possible inclusion in a randomized controlled trial -LRB- RCT -RRB- evaluating several different sexual risk reduction programs . Screening criteria for the RCT included : age 18 or older ; not HIV positive ; and sexual behavior -LRB- e.g. , unprotected sex , multiple partners -RRB- that put them at risk for contracting an STD in the past 3 months . This study used baseline data from the RCT , prior to the receipt of the interventions . Baseline data were available from 1265 eligible participants . Data from participants who refused to answer sexual abuse -LRB- n = 12 -RRB- or demographic -LRB- n = 1 -RRB- questions , were inconsistent in reporting their sexual behavior -LRB- n = 5 -RRB- , were outliers on sexual behavior data -LRB- n = 30 ; defined as having a studentized deleted residual > 4 -RRB- , or were recruited in error -LRB- n = 1 -RRB- were eliminated . Outliers on sexual behavior data were excluded because these individuals likely were members of an extremely high-risk population that merits separate investigation -LRB- Wegener & Fabrigar , 2000 -RRB- . Overall , the sample was 46 % female -LRB- n = 557 -RRB- , 65 % African American -LRB- n = 785 -RRB- , and 24 % Caucasian -LRB- n = 294 -RRB- . The majority of participants were unemployed -LRB- n = 620 ; 51 % -RRB- , had a high school education or less -LRB- n = 762 ; 63 % -RRB- , and had a household income of less than $ 15,000 per year -LRB- n = 686 ; 57 % -RRB- . Most participants were single -LRB- never married ; n = 958 ; 79 % -RRB- ; 75 participants -LRB- 6 % -RRB- were married , and 183 -LRB- 15 % -RRB- were divorced , separated , or widowed . Participants were , on average , 29.2 years of age -LRB- SD = 9.7 -RRB- . Among women , 504 -LRB- 90 % -RRB- reported having sex with only men in the past 3 months ; 53 -LRB- 10 % -RRB- reported having sex with both men and women . Among men , 612 -LRB- 93 % -RRB- reported having sex with only women in the past 3 months ; 31 -LRB- 5 % -RRB- reported having sex with only men ; and 15 -LRB- 2 % -RRB- reported having sex with both men and women in the past 3 months . Procedure Patients who registered for a clinic visit were invited to a private exam room by a trained research assistant -LRB- RA -RRB- , and were asked to answer a series of brief screening questions . The RA explained the study to patients who met eligibility criteria and obtained informed consent . Participants then completed a 45-minute , Audio Computer-Assisted Self-Interview -LRB- ACASI -RRB- that included measures of demographic characteristics , health behaviors and beliefs , and psychosocial functioning , as well as questions about childhood sexual experiences and current sexual behavior . ACASI was used because it optimizes participant privacy -LRB- improving data quality -RRB- while allowing low-literacy persons to participate -LRB- Schroder , Carey , & Vanable , 2003 -RRB- . For the present study , we used data from measures of childhood/adolescent sexual abuse and sexual behavior . After their clinic exam and counseling , participants were paid $ 20 to compensate them for their time . All procedures were approved by the IRBs of the participating institutions . Measures Childhood/adolescent sexual abuse Three items , adapted from Finkelhor 's -LRB- 1979 -RRB- longer survey of childhood sexual experiences , were used to assess sexual abuse -LRB- see Appendix A -RRB- .1 Participants who reported any contact sexual experiences -LRB- including kissing , fondling , giving oral sex , receiving oral sex , vaginal sex , or anal sex -RRB- -LRB- 1 -RRB- before age 13 with someone 5 or more years older or -LRB- 2 -RRB- between ages 13 and 16 with someone 10 or more years older , and those who reported -LRB- 3 -RRB- any contact sexual experience before age 17 involving force or coercion , were classified as sexually abused ; all other participants were classified as not sexually abused . Those who were sexually abused were further categorized according to whether the abuse involved force and/or penetration . Participants who reported a sexual experience before age 17 involving force or coercion were considered to have experienced sexual abuse with force . Sexually abused participants who reported any oral , vaginal , or anal sex were considered to have experienced sexual abuse with penetration . A single , four level categorical variable was created to examine the impact of different abuse characteristics on sexual risk behavior : -LRB- 1 -RRB- no sexual abuse ; -LRB- 2 -RRB- sexual abuse without force or penetration ; -LRB- 3 -RRB- sexual abuse with penetration but without force ; and -LRB- 4 -RRB- sexual abuse with both force and penetration . Too few participants reported sexual abuse with force and without penetration -LRB- n = 39 , 5 % -RRB- to include a sexual abuse with force only category . Current sexual behavior The sexual risk behavior items were developed and tested in previous studies -LRB- Carey et al. , 1997 , 2000 , 2004 -RRB- . Participants were asked to report : the number of male and female sexual partners they had in their lifetime and in the past 3 months ; the number of times they exchanged sex for money or drugs -LRB- lifetime -RRB- ; and the number of times they had been treated for an STD -LRB- lifetime -RRB- . The frequency of unprotected sex was also investigated . Participants were asked to report the number of times in the past 3 months that they had vaginal and anal sex with and without a condom with their : -LRB- 1 -RRB- steady partner ; -LRB- 2 -RRB- other male partners ; and -LRB- 3 -RRB- other female partners . Responses to these items were used to calculate the absolute number and the proportion -LRB- number of unprotected sex episodes/number of protected and unprotected sex episodes -RRB- of unprotected sex episodes in the past 3 months . Table 1Sexual abuse characteristics by genderMen -LRB- n = 643 -RRB- Women -LRB- n = 534 -RRB- Total -LRB- n = 1177 -RRB- n % of menn % of womenn % of totalNo sexual abuse227351823440935Sexual abuse without force and without penetration10016591115914Sexual abuse with penetration only -LRB- no force -RRB- 208321052031327Sexual abuse with both force and penetration108171883529625Table 2Demographic characteristics of sexual abuse groups formed by force and penetrationNo Sexual Abusea -LRB- n = 409 -RRB- Sexual Abuse -LRB- no force or penetration -RRB- b -LRB- n = 159 -RRB- Sexual Abuse -LRB- penetration -RRB- c -LRB- n = 313 -RRB- Sexual Abuse -LRB- force and penetration -RRB- d -LRB- n = 296 -RRB- n % n % n % n % Sex -LRB- male -RRB- 227c , d56100d63208a , d66108a , b , c36Race -LRB- minority -RRB- 269b , c , d66120a , c75279a , b , d89228a , c77Education -LRB- high school or less -RRB- 212b , c , d52100a , c63244a , b , d78191a , c65MSDMSDMSDMSDAge -LRB- in years -RRB- 28.4 d9 .628.79.529.29.830.5 a9 .7 ap < .05 , compared to No Sexual Abuse.bp < .05 , compared to Sexual Abuse , No Force , No Penetration.cp < .05 , compared to Sexual Abuse , No Force , Penetration.dp < .05 , compared to Sexual Abuse , Force , Penetration . Statistical analyses Analyses of variance -LRB- ANOVAs -RRB- were used to determine whether the four categories of sexual abuse -LRB- no sexual abuse ; sexual abuse without force or penetration ; sexual abuse with penetration ; and sexual abuse with both force and penetration -RRB- were associated with later risky sexual behavior . If there was a significant overall effect of sexual abuse , Tukey tests were conducted to determine specifically which groups differed . Demographic variables that differed between groups were controlled for in these analyses . Thus , the ANOVAs included : -LRB- 1 -RRB- demographic covariates and -LRB- 2 -RRB- a main effect of sexual abuse . Continuous outcome variables that were not normally distributed -LRB- i.e. , the number of lifetime partners , the number of partners in the past 3 months , the number of episodes of unprotected sex in the past 3 months , the number of times participants exchanged sex for money or drugs , and the number of previous STD diagnoses -RRB- were transformed using a log10 of -LRB- x +1 -RRB- transformation -LRB- Tabachnick & Fidell , 2001 -RRB- . Unless otherwise stated , analyses associated with these variables used the log transformation . Exploratory analyses were conducted to investigate whether gender moderated the relations between the sexual abuse characteristics and later sexual behavior . ANOVAs were conducted including demographic covariates , a main effect of abuse , and the interaction of abuse and gender . Results Of the 1216 patients who completed the survey , 66 % reported childhood/adolescent sexual abuse -LRB- n = 807 -RRB- . Of these 807 participants who met criteria for sexual abuse , 159 -LRB- 20 % -RRB- reported sexual abuse without force and without penetration , 313 -LRB- 39 % -RRB- reported sexual abuse with penetration , 39 -LRB- 5 % -RRB- reported sexual abuse with force and without penetration , and 296 -LRB- 37 % -RRB- reported sexual abuse with both force and penetration . Because few participants reported sexual abuse with force but without penetration , those participants were excluded from the analyses , leaving a final sample size of N = 1177 . Sexual abuse characteristics by gender are reported in Table 1 . Table 3Sexual risk behaviors of participants who reported sexual abuse with force , sexual abuse without force , and no sexual abuse -LRB- raw data -RRB- No Sexual Abusea -LRB- n = 409 -RRB- Sexual Abuse -LRB- no force or penetration -RRB- b -LRB- n = 159 -RRB- Sexual Abuse -LRB- penetration -RRB- c -LRB- n = 313 -RRB- Sexual Abuse -LRB- force and penetration -RRB- d -LRB- n = 296 -RRB- MSDMSDMSDMSDSexual partners -LRB- number , lifetime -RRB- 31.7 c , d80 .927.6 c , d28 .260.1 a , b211 .564.2 a , b172 .4 Sexual partners -LRB- number , past 3 months -RRB- 2.5 c , d2 .12.72.23.2 a2 .73.5 a4 .0 Unprotected sex -LRB- number of events , past 3 months -RRB- 15.3 c , d24 .317.026.820.7 a30 .122.8 a38 .2 Unprotected sex -LRB- proportion , past 3 months -RRB- 0.680.320.640.330.700.300.670.33 Exchanged sex for money or drugs -LRB- number , lifetime -RRB- 4.9 c , d55 .95.6 d42 .06.5 a , d42 .417.6 a , b , c89 .6 STD diagnoses -LRB- number , lifetime -RRB- 2.4 c , d3 .02.6 c , d3 .23.4 a , b3 .64.0 a , b4 .1 ap < .05 , compared to No Sexual Abuse.bp < .05 , compared to Sexual Abuse , No Force , No Penetration.cp < .05 , compared to Sexual Abuse , No Force , Penetration.dp < .05 , compared to Sexual Abuse , Force , Penetration . Demographic differences Preliminary analyses examined whether any demographic variables were associated with the sexual abuse characteristics -LRB- see Table 2 -RRB- . Sexual abuse was significantly associated with sex , race , education , and current age . Thus , for example , participants reporting a history of sexual abuse were more likely to be less well-educated and more likely to report a minority racial/ethnic identity than nonabused participants . Importantly , sexual abuse involving force and penetration was more likely to be reported by women than by men . All pairwise comparisons for the demographic characteristics are presented in Table 2 . Because of these associations , sex , race , education , and current age were used as covariates in subsequent analyses . Relation between sexual abuse characteristics and sexual behavior After controlling for relevant demographic covariates , sexual abuse was significantly associated with the number of lifetime partners , F -LRB- 3 , 1160 -RRB- = 21.08 , p < .0001 , the number of episodes of unprotected sex in the past 3 months , F -LRB- 3 , 1169 -RRB- = 3.97 , p < .01 , the number of partners in the past 3 months , F -LRB- 3 , 1169 -RRB- = 7.28 , p < .0001 , the number of times sex was traded , F -LRB- 3 , 1153 -RRB- = 14.23 , p < .0001 , and the number of previous STD diagnoses , F -LRB- 3 , 1169 -RRB- = 8.01 , p < .0001 -LRB- see Table 3 -RRB- . Sexual abuse was not significantly associated with the proportion of unprotected sex episodes in the past 3 months . Follow-up Tukey tests showed that , compared to those who were sexually abused with penetration , those who were not sexually abused had significantly fewer : -LRB- 1 -RRB- lifetime sexual partners -LRB- Cohen 's d = .40 -RRB- ; -LRB- 2 -RRB- partners in the past 3 months -LRB- d = .23 -RRB- ; -LRB- 3 -RRB- episodes of unprotected sex in the past 3 months -LRB- d = .19 -RRB- ; -LRB- 4 -RRB- episodes of sex trading -LRB- d = .31 -RRB- ; and -LRB- 5 -RRB- previous STD diagnoses -LRB- d = .22 ; all ps < .05 -RRB- . Similarly , compared to those who were sexually abused with both force and penetration , those who were not sexually abused had significantly fewer : -LRB- 1 -RRB- lifetime sexual partners -LRB- d = .49 -RRB- ; -LRB- 2 -RRB- partners in the past 3 months -LRB- d = .29 -RRB- ; -LRB- 3 -RRB- episodes of unprotected sex in the past 3 months -LRB- d = .21 -RRB- ; -LRB- 4 -RRB- episodes of sex trading -LRB- d = .46 -RRB- ; and -LRB- 5 -RRB- previous STD diagnoses -LRB- d = .34 ; all ps < .05 -RRB- . In addition , compared to those who were sexually abused with penetration , those who experienced sexual abuse without force and without penetration had significantly fewer lifetime sexual partners -LRB- d = .32 -RRB- and fewer previous STD diagnoses -LRB- d = .17 ; both ps < .05 -RRB- . Similarly , compared to those who were sexually abused with both force and penetration , those who experienced sexual abuse without force and without penetration had significantly fewer : -LRB- 1 -RRB- lifetime sexual partners -LRB- d = .34 -RRB- ; -LRB- 2 -RRB- episodes of sex trading -LRB- d = .29 -RRB- ; and -LRB- 3 -RRB- previous STD diagnoses -LRB- d = .30 ; all ps < .05 -RRB- . Finally , those who were sexually abused with both force and penetration reported significantly more episodes of sex trading than those who were sexually abused with penetration -LRB- d = .29 ; p < .05 -RRB- . Because sex trading likely leads to a greater number of sexual partners , episodes of unprotected sex , and STD diagnoses , follow-up analyses were conducted to determine whether penetration was still associated with the sexual behavior outcomes after controlling for sex trading . All effects remained significant after controlling for sex trading -LRB- all ps < .05 -RRB- . Gender as a moderator of the relation between sexual abuse characteristics and sexual behavior To determine whether gender moderated the relation between sexual abuse characteristics and sexual behavior , gender-by-sexual abuse interactions were included in the ANOVAs . Relevant demographic covariates were included . The gender-by-sexual abuse interaction was significantly associated with the number of episodes of sex trading , F -LRB- 3 , 1150 -RRB- = 3.56 , p < .05 . Analyses of simple main effects revealed that , for both women and men , those who were sexually abused with force and penetration reported significantly more episodes of sex trading than those who were not abused , or than those who were abused without force and without penetration . However , for women only , those who were sexually abused with penetration reported significantly more episodes of sex trading than those who were not abused -LRB- all ps < .05 ; see Fig. 1 -RRB- . Fig. 1The effect of the interaction of gender and sexual abuse status on the number of episodes of sex trading Discussion This study investigated whether characteristics of childhood and adolescent sexual abuse -LRB- i.e. , force and type of sexual activity -RRB- were related to adult sexual risk behavior , and whether these associations differed by gender . This research benefited from several methodologic strengths . For example , we sampled a large group of both men and women who reported sexual abuse ; this large and diverse sample allowed exploration of two sexual abuse characteristics and gender differences . We also used psychometrically sound measures and a computer-administered survey , known to result in higher , and presumably more candid , rates of socially stigmatized and sensitive behaviors -LRB- Schroder et al. , 2003 -RRB- . These strengths increase confidence in the validity and generalizability of the results . A key set of findings was that -LRB- 1 -RRB- sexual abuse with penetration as well as -LRB- 2 -RRB- sexual abuse with force and penetration were both related to higher rates of adult sexual behavior compared to -LRB- 3 -RRB- sexual abuse without force and without penetration and -LRB- 4 -RRB- no sexual abuse . This pattern of findings corroborates results from research investigating the mental health sequelae of sexual abuse , which indicate that force -LRB- e.g. , Bulik , Prescott , & Kendler , 2001 ; Rind et al. , 1998 ; Rodriguez , Ryan , Kemp , & Foy , 1997 -RRB- and penetration -LRB- e.g. , Briere & Elliott , 2003 ; Bulik et al. , 2001 -RRB- are associated with worse psychological outcomes ; the current research also adds to the limited body of research suggesting a relation between force and penetration , and later sexual behavior -LRB- e.g. , Cinq-Mars et al. , 2003 ; Fergusson et al. , 1997 -RRB- . The effect sizes for the association between sexual abuse and later sexual behavior were small to medium , indicating that other variables besides sexual abuse account for a large portion of the variance in adult sexual behavior . The latter finding is consistent with the idea that adult sexual behavior is influenced by multiple environmental as well as individual factors -LRB- Smith & Subramanian , 2006 -RRB- . Penetration by itself -LRB- i.e. , without force -RRB- and penetration in combination with force were associated with increased sexual risk behavior relative to those who were abused without force and without penetration , and those who were not abused . The sole difference between the penetration only and the penetration plus force groups involved sex trading , where those who experienced sexual abuse with force and penetration reported engaging in a greater frequency of sex trading , relative to those who experienced sexual abuse with penetration and no force . However , this finding was qualified by a significant gender-by-abuse interaction . Because only a very small number of participants reported sexual abuse with force but without penetration -LRB- i.e. , forced kissing or fondling -RRB- , we were unable to investigate the impact of force only . A somewhat unexpected finding was that the group that reported sexual abuse without force and without penetration did not differ significantly from the nonabused group on any of the sexual behavior outcomes . Future investigation of the relation between sexual abuse and adult sexual behavior might find it fruitful to conduct more fine-grained assessments of the sexual experiences that involve only large age differentials to determine how these experiences are perceived by both men and women , and whether such experiences influence subsequent sexual behavior . It may seem counter-intuitive that individuals who experienced more severe sexual abuse -LRB- i.e. , sexual abuse with force or penetration -RRB- would engage in more sexual experiences than those who experienced less severe sexual abuse ; that is , one might expect individuals who experienced severe sexual abuse to avoid sex because of the negative consequences . However , relative to individuals who experienced less severe sexual abuse , individuals who experienced more severe sexual abuse may use different strategies to cope with their sexual abuse experience -LRB- s -RRB- . Thus , for both men and women , those who experienced more severe forms of sexual abuse may use alcohol or drugs to cope with the sexual abuse , which , in turn , may lead to the exchange of sex for money or drugs , and/or to a greater number of sexual partners and episodes of unprotected sex . In addition , alcohol and other drug use may lead to a greater number of sexual partners and episodes of unprotected sex due to decreased ability to attend to distal concerns , such as acquiring an STD when intoxicated or high -LRB- cf. alcohol myopia ; Steele & Josephs , 1990 -RRB- . Indeed , we have reported previously that substance use is an important mediator of the relation between sexual abuse and risky sexual behavior -LRB- Senn , Carey , Vanable , Coury-Doniger , & Urban , 2006 -RRB- ; future research should explore whether substance use and other potential mediators operate differently for those who experienced different severity levels of sexual abuse . An alternative explanation for the association between more severe sexual abuse and greater adult sexual risk behavior is Finkelhor and Browne 's -LRB- 1985 -RRB- traumagenic dynamics model . This model proposes that one consequence of sexual abuse is traumatic sexualization , in which a child develops maladaptive scripts for sexual behavior , when rewarded for sexual behavior by affection . More severe sexual abuse , such as sexual abuse involving force or penetration , may lead to greater traumatic sexualization . As adults , those who experienced traumatic sexualization may believe sex is necessary to obtain affection from others . Thus , traumatic sexualization may lead to , for example , earlier consensual sex or a greater number of sexual partners -LRB- e.g. , Cinq-Mars et al. , 2003 ; Fergusson et al. , 1997 -RRB- . Another consequence of sexual abuse , according to Finkelhor and Browne -LRB- 1985 -RRB- , is powerlessness , in which a child learns that his or her needs or requests are ignored by others ; the child thus fails to develop self-efficacy to stop unwanted sexual advances . More severe sexual abuse , particularly sexual abuse involving force or penetration , may lead to greater feelings of powerlessness . Perhaps because they lack the interpersonal skills or the self-efficacy to stop unwanted sexual advances , these individuals may be less likely to refuse intercourse with aggressive partners , resulting in more sexual partners . Powerlessness could help explain findings linking more severe sexual abuse to more adult sexual risk behavior -LRB- e.g. , Cinq-Mars et al. , 2003 ; Fergussion et al. , 1997 -RRB- . In this regard , Kallstrom-Fuqua , Weston , and Marshall -LRB- 2004 -RRB- found that sexual abuse severity had an indirect effect on maladaptive relationships , mediated through powerlessness ; thus , having many sexual partners could be a consequence of difficulty forming close relationships . Further research is needed to examine whether the sexual abuse characteristics investigated in this study are associated with Finkelhor and Browne 's -LRB- 1985 -RRB- traumagenic dynamics . Another finding yielded by this study is that abuse characteristics were associated with different outcomes for men and women . For men , only abuse with both force and penetration was associated with a greater frequency of sex trading , whereas for women , abuse with penetration , regardless of whether or not force was involved , was associated with more sex trading . In the current cultural context , young males may view sex with an older woman as masculine and mature , rather than abusive . Males , therefore , may tend to view only experiences involving force or coercion as abusive . Women , on the other hand , may be more likely to view intercourse with an older individual as abusive , regardless of whether or not force was involved . This idea is supported by meta-analytic findings that boys ' reactions to sexual abuse were less negative than were girls ' reactions -LRB- Rind et al. , 1998 -RRB- . Different perceptions of whether or not the experience was abusive may lead to the use of different coping strategies . These results should be interpreted mindful of the limitations of the study . One limitation involved the brevity of the sexual abuse assessment . Use of a brief survey allowed us to obtain a large and diverse sample , but limited the richness of the data collected . The survey did not assess other aspects of sexual abuse , such as duration , frequency , and relationship to the perpetrator , which may be important correlates of later outcomes -LRB- e.g. , Banyard & Williams , 1996 ; Briere & Elliott , 2003 -RRB- . In addition , these brief questions did not allow for assessment of reactions to the sexual experience ; many participants , especially those who did not report force or coercion , may not have considered themselves sexually abused , but may have viewed these sexual experiences as inconsequential or even consensual . Future research , involving mixed qualitative and quantitative methods , might help to elucidate the empirical relations observed in the current sample . A second limitation involves the correlational nature of the data . Clearly , such data limit causal inferences , although given the temporal sequence of childhood/adolescent sexual abuse and adult sexual behavior , the limits may be less concerning in this context . Nonetheless , we acknowledge that unexplored variables that are related to both sexual abuse and greater sexual risk behavior -LRB- e.g. , more adverse childhood experiences ; Dong , Anda , Dube , Giles , & Felitti , 2003 -RRB- should be included in future investigations of the sexual abuse -- risky sex relation . It is important to recognize that participants in this study were recruited from a sexually transmitted disease clinic , and were included because they were currently engaging in sexual behavior that conferred risk for contracting an STD . The rates of sexual abuse reported in this sample were considerably higher than rates -LRB- i.e. , 15 % for men and 30 % for women -RRB- reported in national samples -LRB- Briere & Elliott , 2003 ; Finkelhor , Hotaling , Lewis , & Smith , 1990 ; Vogeltanz et al. , 1999 -RRB- . In addition to engaging in sexual risk behavior , patients attending STD clinics may differ from the general population in other important ways as well ; for example , patients attending STD clinics often report extremely high rates of alcohol and drug use -LRB- Cook et al. , 2006 -RRB- . Due to the nature of the sample , these results of the present study may not generalize to other populations . These results have implications for both practice and research . Regarding public health and clinical practice , they suggest that a thorough sexual health assessment should include inquiry about the nature of the sexual abuse , particularly whether force was involved and what type of sexual act occurred . Given the likely impact of sexual abuse on sexual risk behavior -LRB- as well as other health outcomes -RRB- , we recommend a more comprehensive approach to sexual health assessment , education , counseling , and/or therapy . Indeed , these findings highlight the need to develop interventions tailored to the unique needs of persons with a history of sexual abuse to promote -LRB- and restore -RRB- sexual health and reduce sexual risk . With respect to research , these findings raise many questions about the conditions under which sexual abuse impairs healthy sexual development and expression , and about the mechanisms by which sexual abuse influences sexual development , behavior , and adjustment . This work will require sophisticated methods and analyses to overcome the limitations of what is inherently retrospective and correlational research .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

sexually transmitted disease
hiv
sexual behavior