J ‘1‘..\\\\ L

 

‘\ C‘ "1355'” ‘ '

3m. 1

1“

       
    
 

L’EB C 1 19?!
“54.32% .0 1 1‘ ‘11
[GRWWV

’ 0.94

 

 

OVERDUE FINES:
25¢ nor do per 1:-

gym:& LIQRARY HATEflgALS':
Place in book "turn to move
charge fro-I circulation records

 

 

THE EFFECT OF PENTACHLOROPHENOL ON

THE REPRODUCTIVE PERFORMANCE IN RATS

BY

Kuo-Chuan Karen Chou

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Dairy Science

1980

ABSTRACT

THE EFFECT OF PENTACHLOROPHENOL ON

THE REPRODUCTIVE PERFORMANCE IN RATS

BY

Kuo-Chuan Karen Chou

Pentachlorophenol (PCP) is widely used as an anti-
fungal agent. There is a paucity of data on the effects
of PCP on reproduction. Experiments were carried out to
determine the effects of purified PCP and an industrial
composite of PCP on reproduction in rats. Dose levels of
0, 0.4, 4 or 40 mg/kg of technical grade pentachlorophenol
and the purified pentachlorophenol adjusted to provide the
similar levels of PCP were administered intraperitoneally
to pregnant Sprague-Dawley rats on days 3, 6, 9, 12 and
15 of gestation. The carrier vehicle was corn oil.

Animals treated with the highest levels of either
chemical showed lower maternal body weight gain and
higher incidence of fetal resorptions compared to the
controls. Survival of the newborn pups from the dams

treated with the high dose level of purified PCP decreased.

Sex ratios of the weaning rats showed that fewer female
offspring survived from PCP treatments than the male
offspring.

The activity of hepatic pyruvate kinase increased
more in the dams treated with purified PCP than those
treated with technical grade PCP.

Study of the growth rate and reproduction of the
offspring from the dams treated with PCP showed no
differences between controls and treated animals.

No gross sturctural anomalies were observed among
the offspring. Although more toxic effects were observed
among the animals treated with purified PCP than with
technical grade PCP, the toxic effects were only observed
when higher dose levels (4 or 40 mg/kg of body weight)
were given. Animals in the environment are not likely to
be exposed to PCP at such high levels.

However, the exception is the effect of PCP on the
activity of hepatic pyruvate kinase. For dams treated
with all levels of PCP, including the lowest level tested
(0.4 mg/kg of body weight), pyruvate kinase increased.
This suggests possible abnormal glycolysis and gluconeo-
genesis in the liver. A future study of PCP toxicity in
animals should emphasize effects on the pathway of

carbohydrate metabolism.

To my husband, Edward,
without whom I would not
have started and could not

have finished this study.

ii

ACKNOWLEDGEMENT

The author wishes to express her deepest appre-
ciation to Dr. Robert Cook, whose guidance and support
made the completion of this investigation possible.

Thanks also are extended to the other committee
members, Dr. Steven Aust, Dr. Lee Shull, and Dr. William
Thomas for their helpful suggestions in completing this
study.

Acknowledgement is made to Mr. Mark Jackson for
his hard work and help throughout the study.

Finally the author is indebted to her parents for

their caring and understanding.

TABLE OF CONTENTS

Page
LIST OF TABLES ..................................... vi
LIST OF FIGURES ..... . ................... ...........vii
LIST OF ABBREVIATIONS ........ '.... ..... ...... ...... . ix
INTRODUCTION0.0.0.0. ........... 0....000000. ....... 0 1
LITERATURE REVIEW.... ............... . .............. 3
Chemical and Physical Properties of
Pentachlorophenol......... .................. 3
Method of Industrial Production in U.S.A ...... 6
Toxicity Problems of Pentachlorophenol
in Humans.... ......... ..... ..... .... ........ 7
Toxicity of Polychlorinated Dibenzo-p-
dioxins and Dibenzofurans...... ............ . 10
The Fate of Pentachlorophenol in the
EnVironment...0.0000.0.0 ..... ... 000000000000 l4
Pharmacokinetics and Metabolism of
Pentachlorophenol in Humans and
Laboratory Animals ........... ... ............ 16

Metabolites of pentachlorophenol....... 16
Elimination of pentachlorophenol....... 17
Protein binding of pentachlorophenol

in the bleed. 0 0 0 . 0 0 0 ..... 0 0 . 0 0 0 . . 0 . 0 18
Effects of Pentachlorophenol on Oxidative
Phosphorylation ....... . ..................... 19
Effects of Pentachlorophenol on Glycolysis
and the Tricarboxylic Acid Cycle ............ 20
Teratogenicity and Fetotoxicity Study of
Pentachlorophenol ........................... 21
OBJECTIVES ......................................... 24

iv

Table of Contents (continued) Page
MATERIALS AND METHODS ...... .... ........... . ........ 25

Reagents...................................... 25
Animals....................................... 27
Experimental Design........................... 27
Parameters Measured........................... 29
Enzyme Assay.................................. 30

Protein Determination.......... ......... ...... 31
Statistical Analysis... ............. .......... 31
RESULT8000000. 0000000000000 coo. 00000000 o oooooooooo o 32
Phase I 0000000000 0.... ..... 00.0.0.0..0. ....... 32

Effects on Peed Intake and Body

Weight Gain during Gestation........ 32
Number of Fetuses, Resorption Sites

Sex Ratios, and Weight of Fetuses... 32
Liver Weight, Protein Content and

Activity of Hepatic Pyruvate

Kinase in the Prepartum Rats.. ...... 36

Phase II ...... ..... ......... .. ................ 39

Postnatal Performance of the

Offspring from Birth to

Eight-weeks Old ..... .... ............ 39
Liver Weight, Protein Content and

Activity of Hepatic Pyruvate

Kinase in the Lactating Rats........ 42
Liver Weight, Protein Concentration,

and Activity of Pyruvate Kinase

in the Pups......................... 48

Phase III.......................... ........... 48
Reproductive Performance of the

Offspring................. ......... . 48

DISCUSSION.. ....................................... 55

CONCLUSIONS ........................................ 67

BIBLIOGRAPHY ....................................... 68

LIST OF TABLES

Table Page
1. Solubility of pentachlorophenol in 100 gm of
solvent in the temperature range of 200 - 30°C ..... 4
2. Physical properties of pentachlorophenol ........... 4

3. Composition of commerical grade and purified
pentachlorophenol from The Dow Chemical Co.,
Midland, Michigan............. ............ ... ...... 3

4. Analyses of the Moore and Firestone composites,
and Monsanto purified pentachlorophenol (ppm) ...... 26

5. Effect of pentachlorophenol on maternal feed
intake and body weight gain from breeding to
day 20 of gestation ................. ..... .......... 33

6. Effect of pentachlorophenol on number of fetuses,
resorption sites, and sex ratio............... ..... 34

7. Effect of pentachlorophenol on the body weight
of fetuses at day 20 of gestation.................. 35

8. Effect of pentachlorophenol on the liver weight
of dams on day 20 of gestation..................... 37

9. Effect of pentachlorophenol on hepatic protein
content and pyruvate kinase (PK) activity in
dams at day 20 of gestation.. ...................... 38

10. Effect of pentachlorophenol on the mortality of
neonatal pups ..... ....... ........ ....... ........... 40

ll. Effect of sex on survival of the pups from dams
treated with pentachlorophenol during gestation.... 41

12. Effect of exposure to pentachlorophenol during

gestation on the birth weight and weaning
weight of the pups ................................. 43

vi

Table Page

13. Effect of exposure to pentachlorophenol during
gestation on the feed intake of the offspring ..... 44

14. Effect of exposure to pentachlorophenol during
gestation on the growth of offspring...... ........ 45

15. Effect of pentachlorophenol on the liver
weight of lactating rats three weeks postpartum... 46

16. Effect of pentachlorophenol on hepatic protein
content and pyruvate kinase (PK) activity in
lactating rats three weeks postpartum. . . ........... 47

l7. Liver weight of the female offspring from the
dams receiving PCP treatments during
gestation ..... ...... ............ .... ........ . ..... 49

18. Hepatic protein content and pyruvate kinase (PK)
activity in the female offspring from the dams
receiving PCP treatments during gestation ......... 50

19. Fertility of the offspring from the dams
treated with pentachlorophenol.................... 51

20. Effect of exposure to pentachlorophenol during
gestation on the reproductive performance of
the Offspring..00000000000...... 000000000 .....000053

21. Implantation rate of the bred rats from the dams
treated with pentachlorophenol during
gestation.... ....... .. ...... .. ....... . ............ 54

22. Summary of the experimental results from The Dow
Chemical Company and Michigan State University.... 56

vii

Figure

l.
2.

LIST OF FIGURES

Page
Chemical Structure of Pentachlorophenol ....... 3
Chemical Structure of Polychlorinated
dibenzo-p-dioxin...... ..... .......... ......... 15
Chemical Structure of Polychlorinated
dibenzofuran ..... ............... ........ . ..... 15
Experimental Design ........ , ................... 28

viii

ADP

ATP

AHH
2,6-DCHQ
HCDD

MFO

NADH

OCDD
PCDDS
PCDFs
PCP

PPm

PPb
2,4,5,-T
TCA

TCBQ
TCHQ

TCH

TCP
Tri-CHBQ
Tri-CP

LIST OF ABBREVIATIONS

adenosine diphosphate

adenosine triphosphate

aryl hydrocarbon hydroxylase
2,6-dichlorohydroquinone
hexachlorodibenzo-p-dioxin
mixed-function oxygenases
nicotinamide adenine dinucleotide,
reduced form .
octachlorodibenzo-p-dioxin
polychlorinated dibenzo-p-dioxins
polychlorinated dibenzofurans
pentachlorophenol

parts per million

parts per billion
2,4,5-trichlorophenoxyacetic acid
tricarboxylic acid
tetrachlorobenzoquinone
tetrachlorohydroquinone
tetrachlorohydroquinone

‘tetrachlorophenol

trichlorohydrobenzoquinone
trichlorophenol

ix

INTRODUCTION

Pentachlorophenol (PCP) is a synthetic compound
first synthesized in the 1930's. Registered as a
pesticide agent, it has been used extensively as a
fungicide, insecticide, herbicide, and antiseptic for
more than forty years. -In the last twenty years, it
has become the most widely used wood preservative in
the United States.

Sixty-nine million pounds of pentachlorophenol
were produced in 1975, eighty percent of which was
used by wood preservative processors. Over twenty-five
million cubic feet of lumber and fenceposts were treated
with pentachlorophenol at 155 wood preservation plants
in thirty-one states. This compound has recently
received attention in the animal industry because of
the increasing usage of PCP treated wood in animal
facilities. Morton Buildings, Inc., the largest farm
building company in the United States, reported that
approximately 15,000 buildings are built with PCP
treated wood every year, and most of these are farm
buildings. Livestock of all kinds housed in these
buildings are either directly or indirectly exposed
to the PCP treated wood.

The potential toxicity of PCP to livestock was

recently brought to the scientific community when a

Michigan farm had high mortality in dairy cows (70/232)
and even higher mortality in newborn calves (200/208).
PCP was found in the tissue of these cows as well as in
several other Michigan herds with poor performance.

The levels in the blood and in the livers ranged from

10 to 1000 ppb. Earlier, a high rate of stillbirths

and increased incidence of death in young pigs had been
reported when sows were placed in wooden facilities

that had received PCP treatment. The above findings
indicate the possible effect of PCP on the reproductive
performance of animals. The contamination of food-
producing animals by PCP also raises questions concerning
potential human health hazards. Consequently, the study
of the effect of PCP on the reproductive performance in

animals is needed.

LITERATURE REVIEW

Chemical and Physical Properties of Pentachlorophenol
Pentachlorophenol is a fully chlorinated phenol
(figure 1). The pure product is white, needle-like
crystals soluble in most organic solvents, oils and
petroleum hydrocarbons with aromatic and olefinic
content (Table 1). The solubility in water is very
low. The physical properties of PCP are shown in
(Table 2). It is relatively stable in heat up to the
boiling point. However, under acidic conditions, there
are considerable losses through volatility when it is
heated. The volatility of PCP with steam at 100°C is
0.167 gm of material per 100 gm of steam at standard

atmospheric pressure.

OH
Cl.Cl
C1 C1

C1

Figure 1 Chemical Structure of Pentachlorophenol

Table l Solubility (gm) of pentachlorophenol in 100 gm

of solvent in the temperature range of

 

20° - 30°C
water 1.4 x 10-3 - 1.9 x 10-
methanol 57 - 65
diethyl ether 53 - 60
ethanol 47 - 52
acetone 21 - 33
xylene 14 - 17
benzene 11 - 14
carbon tetrachloride 2 - 3

 

Table 2 Physical properties

of pentachlorophenol

 

molecular weight

melting point

boiling point

density

vapor pressure

(20° - 100°C)

vapor pressure

(160°C)

ignition temperature (air)

266.36
190°C
293°C

1.85

0.11 x 10’3

5.5 mm Hg

- 0.12 x 10’

3

mm Hg

550°C with formation of HCl

 

Pentachlorophenol is a relatively inert compound and

not subject to coupling or substitution reaction common to

most phenols. It dissociates with strong bases to yield

the corresponding water-soluble salts (Reactionl). The

metallic salt of PCP has very much the same efficacy of

pesticide as PCP. Sodium pentachlorophenate is the most

often specified substitution for PCP when high water-

solubility is preferred.

OH

C1 C1 .NaOH

 

C1 C1
C1

ONa
C1- Cl

0 + H2°
C1 C1

C1

Reaction 1 Formation of sodium pentachlorophenate

PentachloroPhenol is decomposed by strong oxidizing

agents such as nitric acid, and converted to a mixture of

tetrachloro-o- and p-quinones (Reaction 2).

 

OH
C1 C1 HNO
C1 C1

C1

C1 Cl
0 +
C1 C1

 

Reaction 2 Oxidation of pentachlorophenol by nitric acid

Pentachlorophenol can be readily converted to the ether
derivative by reacting with diazomethane or diazoethene.
This property is utilized for its analysis by gas chromato-

graphy.

Method of Industrial Production in U.S.A.
Pentachlorophenol is produced in the industry by

chlorination of phenol (Reaction 3).

 

OH OH
catalyst C1 Cl

0 + 5 C12 : O + 5 ac],
elevated
temperature C1 C1 C1

Reaction 3 Preparation of pentachlorophenol

The chlorination is performed in two steps. In the
first step, the phenol and chlorine mixture is held at
temperature 65° - 130°C until the melting point of the
product is 95°C, when most products are tri- or tetrachloro-
phenols. In the second step, reaction temperature is
maintained at 10°C above the melting point of the product
until the PCP content in the mixture reaches the desired
level. This process is performed substantially at
atmospheric pressure, and 0.0075 mole of anhydrous aluminum
chloride per mole of phenol is used as the catalyst. During
this process, polychlorinated dibenzo-p-dioxins, dibenzo-

furans, and lower chlorinated phenols are formed. An example

of the composition of industrial grade PCP is presented in

(Table 3).

Toxicity Problems of Pentachlorophenol in Humans

Pentachlorophenol has been known to be a human toxicant
for many years. It was used in the 1960's as a fungicide in
some laundry detergents. Misuse of these commercial products
containing PCP resulted in twenty poisonings and two deaths
of newborn infants in a hospital nursery (1). Serum and
urine analysis of one ill infant showed PCP levels of 118.0
and 64.6 ppm respectively (2). PCP residue in the contami-
nated linen ranged from 11.5 to 1950.0 ppm. .

The use of PCP as a wood preserver in building material
has also caused several toxicology problems. In one instance,
a woman became ill and was diagnosed as being poisoned by PCP
in her home (9). In another report, a man was poisoned by
bath water contaminated by PCP leaking from pesticide mixtures
used on roof timber (3). PCP was also used at one time to
preserve soy sauce in Japan, although there was no official
report of poisoning.

Many instances of occupational intoxication due to PCP
have been reported between 1945 and 1965. In a West German
plant, workers in the PCP production department complained
of irritation of the eyes and the upper respiratory passages
(4). Ten to seventeen of the workers developed similar skin
disorders characterized by acne with pustular infection,

furunculus, brown pigmentation, and some cicatrization.

.umumeouuommm mmmelzmmumODCEouzo mom coom mad cm uo mm: >2 nmcwsumuwc o

n
m

.>£Qmumoumsouzo Uflsqfialmmm an ConwEHmumc
.Aomv ..oo Mandamno zoo one an oo~>amc<

 

 

m.c om cousuoNcmpfioouoHnomuoo
m.o om causwoNcmbfipouoHnomusz
m.c om couswoncmnwpouonomxm:
AEQQV mcmustNcwnao
o.H comm saxoflolmaoNcmnAconcazomuoo
m.c mad saonoumnoNcmbfloouodzomummz
m.c q cfixedpamIoNCOQHCODOHzomxm:
mo.o mc.o :onfiplmnoNcmnwoouoHnomuumulm.h.m.N
mcfixofloumsoNcmnao
OAEQQV mowaocmzmcoz
m.o ~.w maocmnmxxocmzm poumcwuoHso pmnmfl:
mc.o ~.o HocmnmouoHSOMHB
>~.o v.v Hocmzmouonomuume
+mm «.mm Hocmzmouo~nomucme
EA». mowaococm
omamwusm ocmum ACMOHCEEOO

 

HoconmouoHsocucmm

 

.cmmpzofiz .ocmacpz ..oo Hmofismno son was some
HocmzmouonomucmM oofimqusm can womum Hmfioumssoo mo cofluamomaoo m magma

These symptoms were first noticed weeks or months after the
start of PCP manufacturing. Serveral workers had neurologic
pain of the lower extremities and others complained of
bronchitis. All workers but one still showed extensive acne
more than one year after the plant stopped PCP production.
PCP levels in blood or urine were not determined.

In 1965 five cases of PCP poisoning were reported in
Winninpeg, Manitoba, Canada (5). All victims worked in the
same wood-processing plant. The first worker, who died five
hours after being admitted to a hospital, had been employed
by the plant for only one week. His job description was to
dip wood into a preservative consisting of 4.1% PCP and 0.9%
other Chlorophenols.

The remaining four cases were nonfatal and had symptoms
of excess weight loss, drenching sweating, polydipsia,
evening fever, and elevated basal metabolic rate. One
complained of anorexia and another vomited several times at
work. Urine PCP analysis of these patients ranged from
2,400 to 175,000 ppb. Urine samples of other people working
in the same general area ranged from 400 to 700 ppb. Blood
samples were not collected.

More than fifty instances of occupational PCP poisoning
have been reported during the early years of PCP mass
production. Thirty cases have been fatal. In the last
fifteen years, chemical and wood-processing plants have

instituted proper precautions such as ventilating working

10

areas and the use of protective gloves and aprons. Few
poisoning cases have been reported since then.

There is still a need, however, to study the effects of
chronic exposure to PCP in occupationally exposed people.
Bevenue et a1. (6) has done extensive studies in Hawaii
where he found urine PCP levels in occupationally exposed
individuals ranged from 0 to 1,840 ppb. Wylli et a1. (7)
reported on the case of six employees of a wood-processing
plant in Idaho where samples of serum and urine were colleted
and analyzed over a period of five months. PCP levels ranged
from 348.4 to 3963.0 ppb in the serum and 42.0 to 760.0 ppb
in the urine. Although no signs of toxicity were mentioned,
PCP concentrations in several urine samples from this study
were similar to those reported in other studies of PCP
poisoning cases. Chromosomal aberrations were studied, but
no significant differences were found compared to the normal

population.

Toxicity of Polychlorinated Dibenzo-p-dioxins and
Dibenzofurans

Polychloronated dibenzo-p-dioxins (PCDDs) are a series
of tricyclic aromatic compounds (Figure 2). Different
isomers are formed during the synthesis of a number of
chlorinated phenolic products. In the industrial grade PCP,
octachlorodibenzo-p-dioxin (OCDD) and hexachlorodibenzo-p-
dioxin (HCDD) are found in the highest levels, while 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD) is not detectable at the

11

level of 0.05 ppm. Analysis of technical grade PCP by
Dow Chemical‘Company is presented in Table 3.

There are few reports on the toxicity of PCDD isomers
other than TCDD. But it is generally believed that all
PCDDs have the same toxic effects and Characteristics as
TCDD. Because more is known about TCDD, this knowledge is
often applied to the study of other PCDD isomers. However.
TCDD is considered more toxic than the others, and is very
often described as the most toxic synthetic chemical known.

The toxicity of TCDD has been associated with terato-
genicity in mice, edema in chickens (8), chlorancne in man
(10), and thymic atrophy and immunosuppression in several
species (8, ll, 12, 13, 14, 15, 16).

Since thymic atrophy and immunosuppression are consis-
tent findings in many species exposed to TCDD, researchers
have focused much attention on the direct causes of these
toxic effects and the relationship between these two.

Reduced production of thymic hormones by thymus
epithilial cells can produce thymic atrophy in animals.
Also zinc deficiency is proved to cause atrophy in several
lymphoid organs (17, 18). With these theories in mind,

Vos et a1. (19) studied the cause of thymic atrOphy in
animals. After injection of thymosin to mice given TCDD,
thymus weight and serum zinc concentrations were measured.
Thymosin treatment did not alter the reduced weight of

thymus, nor were the zinc levels different from controls.

12

In addition, phagocytosis of Lesteria Monocytogenes and the
macrophage reduction of nitroblue tetrazolium were studied.
The data indicated that immunosuppression is not due to a
combined effect on both T-cells and macrophages; thus, an
unknown effect of TCDD on T-lymphocytes is suggested.

Gross and microscopic observations showed liver lesions
caused by TCDD in all the animals studied, including mice,
rats and rabbits. Kociba et a1. (22) noted multiple hepato-
cellular degenerative, inflammatory, and necrotic changes in
the liver of rats given 0.1 or 0.001 ug/kg/day for two years.
These animals had increased mortality and decreased weight
gain compared with controls.

TCDD is also considered as a tumor initiator in rodents
(23, 24). The dose of 0.1 ug/kg/day for two years increased
the incidence of some types of neoplasms, but reduced the
incidence of age-related lesions such as tumors of the
pituitary, uterus, mammary gland, pancreas and adrenal
glands which are usually encountered in the rats.

It is not known whether the toxicity of TCDD is the
result of the action of the parent compound or some metabo-
lites. However, elevated activity of certain drug metabo-
lizing enzymes have been reported in TCDD dosed animals (25).
Beatty et al. (26) observed an inverse relationship between
the activity of the hepatic MFO enzyme systems and TCDD
toxicity by measuring the intraperitoneal LD50 in rats.

He proposed that the most logical pathway of TCDD metabolism

13

be through the hepatic MFO enzyme system if TCDD is
metabolized by mammals.

Kitchin and Woods (58) reported elevated activity of
hepatic microsomal aryl hydrocarbon hydroxylase (AHH) in
rats given TCDD. Administration of protein synthesis
inhibitors, actinomycing D or cycloheximide, prevented
TCDD induction of AHH completely; thus, the increased enzyme
activity is explained by increased enzyme synthesis. AHH is
well known to hydroxylate polycyclic aromatic hydrocarbons
to yield cyclic phenols and quinones as well as carcinogenic
compounds. However, scientists have not been able to
demonstrate metabolites of TCDD either in vivo or in vitro
(27).

Teratogenicity of TCDD has been a serious concern in
environmental toxicology (30). Laboratory studies (31, 32)
indicated that a single dose as little as l - 10 ug/kg is
capable of triggering genital malformation in several
research species. The most consistent effects are intestinal
hemorrhage in rats, cleft palate in mice, and kidney
abnormalities in both species. Chromosome mapping showed
increased chromosome damage in the Vietnamese who worked in
the American defoliation program during the Vietnam.War
(28, 29). At that time 2,4,S-trichlorophenoxyacetic acid
(2,4,5-T) was used as the defoliant in Vietnam. TCDD was

present as an impurity in this product.

l4

Polychlorinated dibenzofurans (PCDFs) are similar to
TCDD in chemical structure (Figure 3). The nature of toxic
responses of PCDFs is also similar to that associated with
TCDD (31, 32, 33). The common toxicity symptoms are

subcutaneous edema, thymic atrophy and liver necrosis.

The Fate of Pentachlorophenol in the Environment

Products from photolysis or microbial metabolism of
PCP have been studied in the soil, aquatic environment, and
flyash in the air. Price and Victor (33) observed a ten
percent degradation of PCP in a waste holding pond; 2,3,5,6-
tetrachlorophenol was the major product. Murthy et al. (34)
reported the formation of lower chlorinated phenols from
incubation of PCP-treated soil. Four products were found,
2,3,S,6-TCP, 2,3,4,5-TCP, pentachloroanisole, and 2,3,6-
trichlorophenol (Tri-CP).

Two PCP contaminated rivers in West Germany were
studied (35). Water samples were collected and analyzed for
PCP and its possible products. Six lower chlorinated phenols
were identified in the samples. The author explained that if
PCP degradation to lower chlorinated phenols was extensive in
the river, water samples taken progressively further away
from the origin of the contamination should result in
different ratios of PCP and degradation products. Because no
changes of the ratios of different phenols were found among
the samples taken from different regions of the river, it was

concluded that there was no degradation of PCP to lower

15

Cl

Figure 2 Chemical Structure of Polychlorinated

dibenzo-p-dioxin

O

 

C1
x

Figure 3 Chemical Structure of Polychlorinated

dibenzofuran

16

Chlorinated phenols in these rivers.

Chlorinated dibenzo-p-dioxins and dibenzofurans were
also present in these rivers. Again, there was little
difference in concentrations of these compounds in the
samples collected from different areas.

A single axenic bacterial culture, KC-3, was shown to
use PCP as growth substrate (59, 60). Reiner (36) investi-
gated the mechanism of PCP degradation by studying metabo-
lites accumulating in four mutant strains of KC-3. Tetra-
chlorohydroquinone (TCHQ), tetrachlorobenzoquinone (TCBQ),
and trichlorohydrobenzoquinone (Tri—CHQB) were accumulated
in three mutant cultures, while 2,6-dichlorohydroquinone
(2,6-DCHQ) was accumulated in the other. In summary, the
possible products for the biodegradation of PCP are lower
chlorinated phenols, chloroanisoles, chlorohydroquinones,

chlorobenzoquinones, and polychlorinated dibenzo-p-dioxins

and furans.

Pharmacokinetics and Metabolism of Pentachlorophenol in
Humans and Laboratory Animals
Metabolites of pentachlorophenol

The metabolism of PCP in rats was studied by Braun et a1.
(37). After a single administration of 100 mg of Cl4 PCP per
kg of body weight orally, urine and blood samples were
collected for eight days. Forty-eight percent of the dose

administered was excreted as unchanged PCP, 10% as tetra-

chlorohydroquinone (TCH), and 6% as PCP-glucuronide.

17

Measurement of PCP and metabolites in blood plasma showed

no detectable levels of TCH, whereas more than 80% of the

radioactivity was due to unchanged PCP. A small fraction

was present as PCP-glucuronide. The comparison of TCH

and PCP-glucuronide concentrations between urine and blood
plasma indicated rapid clearance of these compounds in the
plasma through the kidney.

Metabolism of PCP in humans has been studied in four
male volunteers (38). A single dose of 0.1 mg PCP per kg
of body weight was given, and urine.and feCal samples were
collected for seven days.

The total amount of PCP and PCP-glucuronide in urine
accounted for 74% and 12% of the dose ingested respectively.
Four percent of the dose was recovered as PCP and PCP-
glucuronide in the feces. This is similar to the pattern
of PCP excreted in rats except that no detectable level of
TCH was reported. The unchanged PCP has a half-life of

30 hours in human blood plasma.

Elimination of pentachlorophenol

The patterns of PCP excretion in animals is an important
source of information for understanding the mechanism of PCP
toxicity in biological systems. Results of different
investigators have been somewhat variable. Bevenue et a1.
(6) observed PCP concentration in blood and urine of
occupationally exposed people. After a rapid excretion in

the first ten days after exposure, PCP concentration in blood

18

did not decrease more than 60 - 80% for a long period of
time. Thus, he suggested second compartmental control in
human tissue. Braun et a1. (38) could not demonstrate the
two-compartment system model in study of volunteers.
Instead, a one-compartment open-system model was described.
In the study of pharmacokinetics in rats, differences
between sexes and dose levels have been reported (37).

Male rats receiving 10 mg per kg of body weight showed a
two-compartment open-system model of excretion, whereas
data from the female rats receiving the high dose, 100 mg

per kg of body weight, suggested a one-compartment open-

system.

Protein binding of pentachlorophenol in the blood

Protein binding in the blood has been suggested to
increase the retention time of PCP in animals. Hobin et al.
(39) studied the binding of PCP to albumin of three species:
man, rat, and cow. The data of PCP/albumin was nearly the
same. However, the amount of albumin binding in the plasma
of humans was significantly higher than it was in the plasma
of rats. An unknown factor present in the plasma must play
a role in the albumin binding of PCP. This may explain the
longer retention time and high blood values of PCP in humans
as compared with the data from rats (40, 41).

Hot weather has long been associated with the toxicity
of PCP in humans and animals (5, 42). It is believed that

high environmental temperature can trigger PCP poisoning in

l9

occupationally exposed people. Studying the PCP binding in
plasma or rats, Hoben et a1. (39) found a reversed relation-
ship between temperature and the amount of bound PCP. The
increased toxicity of PCP in hot weather may be explained

by decreased PCP-albumin binding thereby increasing the
amount of free PCP in plasma. It is suspected that PCP in
the free form rather than albumin bound PCP causes toxic

effects in the biological system.

Effects of Pentachlorophenol on Oxidative Phosphorylation

Pentachlorophenol is an uncoupler of oxidative
phosphorylation with the characteristics of biological effect
similar to 2,4-dinitrophenol (43). Incubation of rat-liver
mitochondria with PCP showed increased activity of adenosine
triphophatase (ATP-ase) and increased oxidation of glutamic
acid. However, PCP has no effect on oxygen uptake by the
mitochondria except when high concentrations of PCP were
incubated with the liver mitochondria and the process of
oxidative phosphorylation is completely inhibited.

The radical uncoupling of oxidative phosphorylation in
the tissue is responsible for the increased basal metabolic
rate and hyperpyrexia common in PCP poisoned individuals.
This may also explain why PCP poisoning was usually associated

with environmental temperatures above 80° - 90°F.

20

Effects of Pentachlorophenol on Glycolysis and
the Tricarboxylic Acid Cycle

The uncoupling effect. of PCP in animals interrupts
the link between energy supply and energy utilization.

When fish were exposed to PCP, ATP concentration was decreased
and therefore mitochondrial respiration was stimulated (44,
45, 46). Greater mitochondrial respiration demands more
substrate; thus, the activity of enzymes in the tricarboxylic
acid (TCA) cycle are expected to increase. Kruger et a1.

(47) and Bostrom and Johansson (46) studied PCP poisoning in
fish and showed that PCP increased activity of isocitrate
dehydrogenase and fumarase in the liver.

To meet the requirements of substrate for the high rate
of TCA cycle, one would also eXpect the increased rate in the
glycolysis in PCP poisoned fish. However, Bostrom and
Johansson (46) observed decreased activity of pyruvate kinase
and lactate dehydrogenase when PCP poisoned fish were starved
for 4 days. Their study indicated the possible decrease in
the rate of liver glycolysis. In the starved animals the
rate of liver glycolysis was depressed due to the unavaila—
bility of glucose. Therefore, the rate of oxidation of body
fat may be increased to supply substrates for TCA cycle. In
_normally fed animals one should observe increased rate of
glycolysis to meet substrate requirements for a high rate of
flux through the TCA cycle as explained in the previous

paragraph. The present study was designed to measure

21

activity of hepatic pyruvate kinase as an indication for

the rate of glycolysis in nomally fed rats treated with PCP.

Teratogenicity and Fetotoxicity Study of Pentachlorophenol
There is a paucity of information on reproductive
performance affected by PCP exposure. To our knowledge
only two full papers have been published. They are one by
Schwetz et al. (50) from the Dow ChemiCal Company, and the
other by Larsen et a1. (51) from Scott and White Clinic.
Additionally, there are two abstracts, one by Hinkle (48)
from the Environmental Protection Agency, and another by
Schwetz and Gehring (49) from the Dow Chemical Company.
Hyperthermia is known to have a teratogenic effect on
developing rat fetuses on day 9 through day 14 of gestation
(52). Poisoning by PCP had always shown the elevated body
temperature as mentioned previously. With these two facts
in mind, Larsen et a1. (51) studied placental transfer and
teratogenicity of PCP in rats. When an oral dose of 60 mg
per kg of body weight was given to rats, 0.05 to 0.1% of the
administered dose was found per gram of fetal tissue. Body
temperature increased about 0.5 to 0.8°C in the animals
receiving PCP; however, these were considerable lower than
the temperature known to produce teratogenic effects. No
significant increasing incidence of resorption and malfor-
mation were observed by giving a single oral dose of
60 mg/kg at day 8, 9, 10, 11, 12 or 13 of gestation. The

compound used was 99% pure PCP.

22

Schwetz and Gehring (49) reported the evidence of
fetal toxicity effects by giving daily PCP treatment from
day 6 through 15 of gestation. Subcutaneous edema, dilated
ureters and minor anomalies of the skull, ribs, vertebrae
and sternebrae in pups were observed at an incidence which
increased with increasing dose levels. The no effect level
was 5 mg/kg/day.

Hinkel (48) gave 1.25 mg/kg or 20 mg/kg of PCP daily
to hamsters from day 5 to 10 of gestation. Fetal deaths
and/or resorptions were observed in 3 of 6 test groups.
Concentration in entire fetuses was closely correlated to
the concentration of PCP in maternal blood.

To determine the possibility that the nonphenolics in
the commercial preparation of pentachlorophenol might be
the cause of fetotoxic effects, Schwetz et a1. (50) studied
the comparison of toxic signs produced by purified and
commercial grade pentachlorophenol in rats (see Table 3 for
composition of purified PCP used). Maternal weight gain
was decreased among dams treated with 30 or 50 mg/kg/day
of either technical or purified PCP. Fetus resorptions
increased from 4.2% in the controls to 27% in 37.7 mg/kg of
commercial PCP treated rats and to 50% in 50 mg/kg treated
rats. Purified PCP increased the resorptions to a greater
extent; 100% in the 30 mg/kg and 97% in the 50 mg/kg
treated rats. The ratios of male fetuses to female fetuses
,were increased with increasing amounts of PCP. Fetotoxic

signs observed were similar to the study reported previously

23

(49). Purified pentachlorophenol caused the same forms of
anomaly, but showed slightly more toxic effects than the
commercial grade. The no-effect dose level of commercial
grade PCP was 5 mg/kg/day; purified pentachlorophenol at
the same level showed a significant increase of delayed
ossification of skull bones.

In addition to the lack of information on the effects
of PCP on the reproductive performance, none of the studies
published have observed the postnatal function in the
offspring from the dams receiving PCP treatments. The
influences of the xenobiotics may not be limited in their
effect to intrauterine life, but also exert their effects
after birth. It is well known that in humans, only 10% of
the abnormalities may be observed in neonates, while others
will be discovered in the later developmental span. We also
feel that the impairments of biological functions of the
animals are just as important as the structure anomalies.
An individual may survive well and appear normal in the
postnatal period but belong to the categories of functional
deficit in later life. Therefore, it is necessary to

determine postnatal performance.

(l)

(2)

(3)

OBJECTIVES

Compare the effects of technical grade PCP and
purified PCP on rat reproductive performance.

Study the mortality, growth rate and reproductive
performance of the offspring from the dams treated
with PCP.

Study the effects of PCP on the activity of hepatic
pyruvate kinase in pregnant rats, lactating rats

and offspring.

24

MATERIALS AND METHODS

Reagents

The technical grade PCP and purified PCP were gifts
from Monsanto Industrial Chemical Co., St. Louis, Missouri
in 1977 (Table 4). The technical grade PCP was a composite
of industrial grade pentachlorophenol from Reichhold, Vulcan,
and Monsanto chemical companies. The composite was produced
from the same lots as composites for Dr. David Firestone and
Dr. John Moore of NIEHS, but not at the same time. It was
also part of a composite prepared for the National Cancer
Institute. Analytical data was provided for the two indus-
trial composites prepared for Drs. Moore and Firestone.

Adenosine diphosphate (ADP), lactic dehydrogenase,
phosphoenolpyruvic acid (crystalline, trisodium salt), NADH,
and triethanolamine - HCl buffer (pH = 7.5) were purchased
from Sigma Chemical Co., St. Louis, Missouri. Monobasic
sodium phosphate, dibasic sodium phosphate, potasium chloride
and magnesium sulfate were purchased from Mallinckrodt, Inc.,
St. Louis, Missouri. Sodium chloride was purchased from
Mallinckrodt, Inc., Paris, Kentucky. Ethyl ether was
purchased from J.T. Baker Chemical Co., Phillipsburg, N.J..
Corn oil was manufactured by International Inc., Clinton,

Iowa.

25

26

pwusmmme uoz n

.00 mHmOMEmco HmfluumSUCH oucmmcoz >2 pmn>amcc m

 

couswoNcmaapouo~nofiue

 

 

nun mo.o v ~o.o 6:6 aexcaeumnoucmaacouoHBUHHe

cmusmoNcmanouonomuume

all N.o e.c ppm cawapualoNconfipouonomuuwe

sensuoucmnflpouonochmm

nII m.o H.H pcm :wxofipImIONcmnflpouOHzomucmm

copsmoncmnflpouoHnomxmz

~e.o «.ma ~.GH new :HxOSCuduoNcmnAconcasomxmz

cmuzmomcmnwpouoHnomusz

m.m Rea LNG can saxomeua-oNcmnaoouoagomuamz

o.o am mad cmusu0~amnaoouonomuoo

m.~ cued cave aonficnduoNcmnAnchoHaomuoo

m.¢ cm on mcmucmnouoHnomxmz
OUQOmCOZ OCOumOHfih .HQ thOS ..HQ

mom cmampusm mom chewedsoo

 

.mAEQQv HocmcmouoHcomucmm pmwwfiusm
Oucmmcoz pcm .mmufimomeoo ocoummufib pcm Choc: may no mmm>amc< v mange

27

Animals

Eighty Sprague-Dawley rats from the Lab Animal Care
Service of Michigan State University were used in this study.
Five female rats, weighing approximately 300 gm, were caged
together with a male breeder. Copulation was checked every
day at 8:00 a.m.. The day on which sperm was first seen in
a vaginal smear was considered day 0 of gestation. After
breeding the animals were then housed individually in the
animal laboratory which was controlled for temperature
(74° - 76°F), and light cycle. Water and commerical labora-

tory rat chow were provided ad libitum throughout the study.

Experimental Design (FIGURE 4)

This experiment is divided into three phases. Phase I
was the study of reproductive performance of the pregnant
rats treated with either purified PCP or technical grade
PCP. Thirty-nine bred rats were assigned randomly into seven
groups. Rats in each group were treated intraperitoneally
with 1 ml of corn oil containing different levels of either
purified or technical grade PCP on day 3, 6, 9, 12 and 15
of gestation. The rats in the control group received only
corn oil. Dose levels are 0.4, 4 or 40 mg of technical grade
PCP per kg of body weight and 0.34, 3.4 or 34 mg_of purified
PCP per kg of body weight. These levels are designated as
low, medium.and high in this paper. All the rats were
sacrificed on day 20 of gestation and investigated for the

activity of hepatic pyruvate kinase. Data were also collected

28

cmfimma Hmucoeflummxm v uneven

H nnnnnnnnnnnn HHH swarm a uuuuuuuuuuuuu H H1IL nnnnnn HH amaze uuuuuuuuuuuuuuuuuu H
H nnnnn sewucummm lllll H H IIIII coflumumom ((((( H
pHo pao
AN o mxom3 NH mxmoz m «w a
wee >ma mesa mesa awn ago

 

 

       

 
 

.ccvccaaaaacaavaecaéa.
.ufifluusuuuuuuuuusuxqfi
......................

ace2vacccavavavvvvvavrvvaappprvvvpa
.aqfianuaaaqqqcauqfiuqxquxfisqfiaqxafisu
............. .........o...........

 

    

 

 

 

 

      

 

   

 

 

 

 

 

 

 

  

 

 

+ + meWEwmwnw mum
coHufiusuumm
meadows
mcapwmun
onwmwuomm
Hsuunsu: uuuuuuuuuuuu H amaze nnnnn u ....... H
Hull! lllll In: sssss In cofiumummm nnnnn IllnIIIIH
mm cm ma «A m m m o woo

mucosacouu mum
mowmquomm

29

on the fetuses at this time.

Phase II of this experiment was the study of the
development of the pups from the PCP treated dams. Thirty-
seven bred rats were assigned randomly into seven treatment
groups and received the same treatments as decribed in
Phase I, except that the animals were allowed to give birth
and nurse the pups until weaning.

In Phase III, the reproductive performance of the
offspring from dams treated with PCP was studied. Two male
and two female offspring from Phase II experiment were
selected randomly from each litter at 12 weeks of age.
Copulation was allowed by caging one female with one male
from the same treatment but from a different litter. Then

the animals were sacrificed and the fetuses were recovered.

Parameters Measured

In Phase I, animals were observed daily for signs of
toxicity. Body weight and feed intake were recorded at
3-day intervals. On day 20 of gestation, animals were
sacrificed and the activity of hepatic pyruvate kinase was
investigated in the dams. The uterus was opened for
recording the number of fetuses (litter size), position of
live, dead, and resorbed fetuses, sex ratios, weight, and
external anomalies of the fetuses. In this study, sex ratio
was defined as the number of males relative to the total

number of fetuses.

30

In Phase II, the number of pups born alive or dead and
data for 3-day, l-week, 2-week and 3-week survivals were
recorded. Body weight of the pups was taken at 7-day
intervals from birth to 8 weeks old. Feed intake was taken
from.weaning (3 weeks old) to 8 weeks old at 3-day intervals.
The activity of hepatic pyruvate kinase was investigated for
the dams at 3 weeks postpartum and for the pups at 8 weeks.

In Phase III, copulation was taken as an indication of
a complete estrual cycle for female rats; therefore, data
were recorded showing the number of days the female and
male were caged together until the sperm was first seen in
vaginal smear. This information was also applied as the
indication of the libido for the male rats.

After the female rats were bred, body weight was
recorded at 7-day intervals from day 0 to day 21 of gestation.
Then the animals were sacrificed and the fetuses were observed
for sex ratios, weight, and external fetal anormalies. The
number of corpora lutea was recorded as an indication for

ovulation.

Enzyme Assay

Activity of hepatic pyruvate kinase was determined using
the method of Bucher and Pfleiderer (20). In this method the
total amount of pyruvate kinase is measured including the

activity of the enzyme in both forms, active and inactive.

31

Protein Determination
Protein was determined by the method of Lowry et a1. (21)

using a Coleman Junior Spectrophometer.

Statistical Analysis

The differences between control and test values for each
of the parameters measured was determined using analysis of
variance followed by the Dunnett-test. The specific compari-
sons of the effects of technical grade PCP with that of
purified PCP were analyzed using designed non-orthogonal

contracts and Bonferroni t-test.

RESULTS

Phase I:

Effect on Feed Intake and Body Weight Gain during
Gestation (Table 5)

Total feed intake was not changed by PCP injection in
any treatment groups. However, animals receiving high doses
of purified PCP gained less weight than the controls. For
the other treatment groups there was no difference of weight

gain during gestation.

Number of Fetuses, Resorption Sites, Sex Ratios (Table 6)
and Weight of Fetuses (Table 7)

A mean of 4.8 fetuses per litter was recovered from the
dams receiving the high dose level of purified PCP, while
12.8 fetuses were recovered from the controls. These two
values ar statistically different at the level of 0.01..
There was not enough evidence to tell that either the medium
or low dose level of purified PCP treated groups had different
litter sizes from the controls. None of the technical grade
PCP treatments affected litter size.

The number of fetuses per litter for the three purified
PCP treated groups was pooled to compare with the pooled data
of technical grade PCP treated groups. Treatment with
purified PCP resulted in smaller litter size than with

technical grade PCP (p < 0.05). Decreased litter size may

32

33

.mooc V Q sGDHM> HOHUCOO 0:8 EOHW UCQHQHMHQ ewHuEMUHWflC—wflmw

 

 

o
.Ho.ouv e .m:~m> Houucoo onu Scum ucmummmflo aaucmofiuwcmwm 2
.coHumumom mo ma .NH .m .w .m mamp co HMO cuoo Cw mH pmuoumficflspc m
H.m a.mmm m.a «.mma w.md a.mmv m oo.ov
a.ma m.m~v h.oa o.~na a.ma m.omv v oo.v
o.o o.aae o.o o.HvH o.o a.mmv a ov.o
compo HmoHccoou
h.ca «.mmm o.vH h.ve w.ma o.~vv m oo.vm
~.mH c.5Hv e.m a.mwa a.mm m.mhv e ov.m
m.ma ~.nav m.oa o.HmH m.vm ~.mhv v em.c
OOHMflHSQ
.HoconmouoHcomucom
~.cH o.omm v.oH v.~ma ~.m~ ~.vmv m Houucou
.m.m m .m.m M .m.m M
xsmo\mx\me.
.Emv A55. Asmv mamaficm mmOC 0cm
unmflm3 upon dough Ceca ucmwmz mxmucw come mo .02 mHmHHmume Emma

 

.cofiumummm mo om amp ou mcflpomun
uzmwOB apoa pcm mxmucfi comm Accumuma co HocmnmouoHnomuch mo uommmm

Eoum seem
m manna

34

 

 

.mo.ouv a .mSHm> Houucoo mcu Scum ucmummuflp >ADCMOMMflcmfim p
.mc.o V a .mum mpmum Hmoflccomu sue: pmumouu mameflcm may Scum dump pmaoom
0;» can» Demumuuac ma non saucepan rues cmummun mamsacm 0:0 some 6066 coaoom on
.Hc.ouv a .msam> Houucoo may Scum ucmumwmflp zaucmofluwcmam m
ha.c mm.c >.o v.H N.H 0m.HH oo.oe
qa.o Hm.o ~.o «.0 m.c 0m.ma oo.v
oc.o mo.o o.c c.c o.o oo.ma ov.o
wpmuq ducacsomu
pea o mm o o N co m m H pom v co em
wH.c em.o ~.c ~.c ¢.o am.HH ov.m
mm.c mm.o o.~ ~.H o.H bm.aa vm.o
emauauaa
.Hocmnmouonomucmm
ea.o am.o ~.o ~.o. k.o a.mfi m Houucoo
.m.m M .m.m M .m.m M
mmuwm A>m0\mx\mav
ofiumu xmm coHumuommu momsumu mumuuaa om0p pcm
mo .02 mo .02 we .02 Hawumums umme

 

.ofiumu xmm
pcm .mmuflm :OquHOmOH .mmmsumu no Lopez: :0 Hocmnmou0Hcomucmm mo uomumm o mange

35

Table 7 Effect of pentachlorophenol on the body weight
of fetuses at day 20 of gestation.

 

 

 

Test material No. of Body weight of fetuses
and dose litters
(mg/kg/day) male (gm) female (gm)
2 5.5. R 3.3.
Control 5 5.8 0.1 5.7d 0.1
Pentachlorophenol,
purified
0.34 4 5.8 0.2 5.6 0.2
3.40 4 6.0 021 5.8 0.2
34.00 4 s.2° 0.2 4.4°°0.2

technical grade

 

0.40 l 5.9 0.0 5.5 0.0

4.00 4 5.9 0.0 5.6 0.1

40.00 5 5.7 0.1 5.2 0.2
a

Only two litters were observed for the female fetus
body weight, because the other two litter in this
group had all male fetuses.

Significantly different from the control value, p <:0.05.
Significantly different from the control value, p <=O.l.

Significantly different from the pooled data of female
fetuses from all six PCP treated groups p < 0.1.

O

36

explain the decreased maternal body weight gain in the same
treatment group.

The sex ratio of the fetuses was higher among the dams
treated with the high dose level of purified PCP compared
with controls. This is not statistically significant because
of the high resorption rate in this group and the small
number of observations. The number of resorption sites in
the high dose of purified PCP treated group (mean = 5.0) was
larger than that of controls (mean = 0.2), p‘< 0.05. However,
the evidence of increased resorption sites in the other
treatment groups was not very strong.

Both female and male fetuses in the group treated with
the high dose level of purified PCP had decreased body weight.
However, the evidence of decreased body weight for the male
fetuses (p < 0.1) was not as strong as it was for the female
fetuses (p < 0.05). The body weight of the male fetuses in
the other groups was not changes by the PCP treatment, while
pooled information of the female fetuses from six PCP treated
groups showed decreased body weight compared to the controls

(p < 0.1).

Liver Weight (Table 8), Protein Content and Activity of

Hepatic Pyruvate Kinase in the Prepartum Rats (Table 9)
Liver weight was not affected by any of the PCP treat-

ments. No changes of liver protein content of specific

activity of pyruvate kinase were observed in any of the

37

Table 8 Effect of pentachlorophenol on the liver
weight of dams on day 20 of gestation.

 

Test material

 

and dose Liver weight (gm)
(mg/kg/day)
I 5.2.
Control 14.7 1.0
Pentachlorophenol,
purified
0.34 17.5 1.4
3.40 15.7 0.6
34.00 14.6 0.5

technical grade

 

0.40 15.9a 0.0

4.00 16.2 0.9

40.00 17.0 1.0
a

One observation in this group.

38

.msmmflu Dos wo em you ewe mom pace 5

 

p
.cflmaoum mo Em Hoe cfiE mom mace s o
.Qsoum menu CH c0wum>ummbo 0:0 n
.mmumcmmoeo: um>wa no as you me o
c.a a.ma No.0 cm.v~ H.m v.5m oc.ov
o.~ m.wa mo.c mm.mm v.m a.mm co.v
o o bu a oo o bem.m~ o.o em mm av o

momma Hmofic500u

II . II II oo.vm
m.o H.MH Ho.o c~.~N v.~ v.mm ov.m
N.H v.ma No.c ec.~m c.m m.mm vm.o
pmfiMAusQ

.HocmCQOHOHcomucmm

 

~.c a.ma Ho.o ma.- ¢.H a.mm Houucoo
.m.m M .m.m m .m.m M

60>AH so no x~e\mev x>6o\mx\osv

no Scum Mom >ua>wuom ucmucoo choc pcm

pxm mo mudc: oofiwaommm mcfimuoum Hmfiuoume Dame

 

.cofiumummm mo om >mp um mumh :fl >uq>wuom Anne macawx
mum>su>¢ pcm acoucoo caououm Oeummo; co HococmoungomucoQ mo uommum a canoe

39

animals that received different levels of technical grade
PCP or in those rats that received the medium or low dose
levels of purified PCP. The protein content and activity of
pyruvate kinase were not determined in the liver homogenates
of the rats treated with the high level of purified PCP.
Pyruvate kinase activity per gram of liver was not

significantly different among all the treatment groups.

Phase II:

 

Postnatal Performance of the Offspring from Birth to
Eight-Weeks Old

The mortality (Table 10) of pups from dams treated with
purified PCP tended to be higher than the pups from the dams
treated with technical grade PCP from birth to day 3 after
birth (p <=0.12). The mortality of pups from day 3 to day 7
and from day 8 to day 14 after birth was higher among the
groups receiving the low dose level of technical grade PCP
and the high dose level of purified PCP. However, the
statistical evidence is not strong, because the high mortality
was observed only in one of the five or six litters in each
treatment group.

The number of male pups surviving per litter at weaning
(Table 11) was not affected by either chemical, but the
number of female pups surviving in the group of dams receiving
high doses of purified PCP was significantly decreased
(p <10.01). The pooled number of female pups surviving from

the three groups treated with purified PCP was less than that

40

.ma.ouv Q .mom momma Hmoficnoou mo msoum scum

dump pmaoom ozu cmzu acoumwmwp we mum pm«MMusm mo macho Eouu dump pmaoom

 

 

 

co
.ucmsummuu 05mm on» c« muouuwa 0:» mo mco cw pm>ummno mane mo gamma 2
.uouuad mom sauce «0 popes: may no omnum>¢ m
o o co e co co
o o Co v co v
m o am e m A no A co m ow o
mpmum Aguacnomu
m 0 pm o N c an o v o Do A o co em
N.H ow.H m ov.m
o 0 0o m vm.o
poflmwusm
Hocmzmouodsomucme
o o o w Houucou
.m.m m .m.m M .m.m M
as oo o soo A on a sot m soo on tough .soo\ox\oec
mumuuwa OmOp 0cm
mcumon mo .02 mo .02 aneumpme umme

 

.mmsm Hmumcomc mo aufiamuuoe ozu co HocmzmouoHcomucmm mo uomumm ca mange

41

.Hc.c V Q .mum momum HCOMCCOOH mo mesoum Eoum pump
pmaooa on» can» uCOHOMMAC ma non pmfimfiusm mo mesoum Scum pump pmaoom

 

 

 

co
.ac.ouv Q .maouucoo any Scum ucmummmflp haucm0emwcmfim 2
.mmsm mo Hones: Howey on» on m>wumamu moans Mo “03552 c
~o.o cm.o m.o p~.v m.o h.o oo.cv
vo.c Nv.o c.H pm.h m.a m.m oo.v
oo.c av.o c.H po.m b.c m.v ov.o
mpmum Hmowccomu
me o Ema o m.o Unm.a o H m m co.vm
mo.o mm.c o.H 0m.v h.o N.o ov.m
vo c mm.o c.a om.m o.H o.> vm.o
pmfimflusm
.HocmcmouoHnomucom
Ho.o me.o m.o ~.m v.o o.m Houucoo
.m.m M .m.m M .m.m M
mumEmm mane Aamp\mx\msv
moflumu xom omen 0cm
mcflcmw3 um mca>fl>usm mesa mo .02 Hmflumume umme

 

.COMumummm mcwusc Hocmnmouonomucmm
zuwz cmumouu mEmp EOHu mean on» no Hm>w>upm co xom mo uommwm Ha mange

42

from the three groups treated with technical grade PCP
(p < 0.12) .

The sex ratio of the pups at weaning was significantly
higher for the high dose level of purified PCP treated group
than the other groups.

The combined body weight of male and female pups at
birth and at weaning showed no significant differences among
all the groups (Table 12).

The total feed intake of the male or female pups from
weaning to eight weeks old showed no significant differences
from the control value (Table 13). However, the feed intake
of the female pups from the high dose level of purified PCP
treated group was significantly less than that from the high
dose level of technical grade PCP. The body weight of the
pups taken weekly from four weeks to eight weeks old was the

same among all the groups for either sex (Table 14).

Liver Weight (Table 15), Protein Content and Activity of

Hepatic Pyruvate Kinase in the Lactating Rats (Table 16)
Liver weight was not changed by any PCP treatments.

Pooled data of protein concentration and unit of pyruvate

kinase per gram of liver were higher from the purified PCP

treated animals than that from the technical treated ones

(p <:0.12 and p ‘<0.05, respectively). However, none of

the data for individual treatment was statistically signifi-

cantly different from the control values. The specific

activity of pyruvate kinase was similar in all of treatment

groups.

43

Table 12 Effect of exposure to pentachlorophenol during

gestation on the birth weight and weaning weight
of the pups.

 

Test material

 

and dose Birth weightab weight at weaningab
(mg/kg/day) (gm) (gm)
2' 5.12:. ii 5.5.
Control 6.93 0.15 44.3 3.4
Pentachlorophenol,
purified
0.34 6.88 0.12 41.8 1.1
3.40 6.48 0.19 46.7 2.3
34.00 6.48 0.10 46.7 4.2
technical grade
0.40 6.58 0.32 42.6 2.8
4.00 6.83 0.12 39.5 1.6
40.00 6.65 0.12 46.3 1.7

 

a

Data is reported as the average of the combined body

weight of male and female pups per litter.

b

No statistical significance was observed.

44

Table 13 Effect of exposure to pentachlorophenol during
gestation on the feed intake of the offspring.

 

Test material

Feed intake (gm)

 

 

and dose from 3 weeks to 8 weeks old
(mg/kg/day)
male female
2 5.5. E 5.5.
Control 638 14 520 5
Pentachlorophenol,
purified
0.34 612 16 510 7
3.40 625 10 503 10
34.00 609 24 471a 41
technical grade
0.40 607 15 508 10
4.00 624 14 511 29
40.00 637 14 538b 14

 

ab

Significantly different from each other, p < 0.1.

45

.mmsoum ucwEumwuu ozu Ham GCOEC mocoofimwcmfim HMOAUmwumum 0: ma ouoze

 

 

 

 

m
H.@ ,m.¢m~ v.9 m.mm~ m.a m.mb m.m a.mm oo.ov
v.5 m.>m~ c.- m.ew~ o.m m.o> m.m N.vh oo.v
m.v ~.emH a.ma v.¢o~ o.v v.vh n.m ~.om ov.o
momma Hooficzoou
h.v o.mcm H.oH mucmm m.m m.we o.HH a.mm oo.vm
m.m c.maa v.m v.mn~ ~.N o.~m 5.5 m.mm oq.m
m.v o.~m~ m.h N.H>~ m.a m.Hh m.N m.me vm.o
ooamaooo
.HocmcmouoHnomucom
v.v m.mo~ v.m m.mm~ c.m m.mh h.m v.vm Houucoo
.m.m m .m.m m .m.m m .m.m m
mmamsmu moans amamemw dodge
Asoo\ox\osd
Aewv Asmv mmoo ocm

oao mxooz m an unmfim3 zoom

oHo mxomB e um Davao: zoom

Hmfiuoame umme

 

any :0 coflumummm mcfluso

.mcwummmwo
HocmszHOHcomucmm o» musmomxm

mo :u3oum

HO UOQNMM VA OHQOE

46

Table 15 Effect of pentachlorophenol on the liver weight
of lactating rats three weeks postpartuma.

 

Test material

 

and dose Liver weight (gm)
(mg/kg/day)
Ti 5.5.
Control 20.7 1.4
Pentachlorophenol,
purified
0.34 19.1 0.5
3.40 19.3 ‘1.0 '
34.00 21.5 1.9

technical grade

0.40 18.9 1.4
4.00 21.4 1.1
40.00 20.3 ~1.2

 

a There is no statistical difference among the treatments.

47

.cms me omueawu: mmummumasm mo mHOE a a
.mc.o V Q .mom momma Hmomcnomm an“: ommmmmu mHmecm 50mm mmmo

omHOOQ may cmnm acmmmmmmo mm mom omawmmsm :mmz ommmmmu mHmEmcm Eomm mumo omaooe um
.NH.o V a .mom momma amOmccomm cum: omummmu mHmEmcm Eomm mumo

omaooe mcu cmnu ucmmmumwo mm mom omflmmmsm :uw3 omummmu mHmecm Eomm mumo omHoom om

 

 

 

.cwmuoma mo Ea mmm :me mma mHOE : a
.mmumcmaoeoz mm>mm mo HE mmm cmmuoma mo aE m
mu mama vm.o mvm.m mo.c om.ma v.m oo.mm oc.ov
mm meow mo.m umN.HH mo.c om.- m.~ oh.om oo.v
Hm meow mm.H mom.o~ no.9 oc.m~ m.H oh.w~ oe.o
momma Hmomczomm
Hm mmmm we.c mam.mm mo.o ma.v~ o.m mo.mm oo.vm
Hm mamm oc.c mvw.qa ~c.c ma.m~ m.m 0v.mm ov.m
ha meow , mo.m mv~.~H Ho.c wh.om o.~ mo.mm vm.o
oommmmoo
.HocmzmomoHnomucmm
mm mNN wm.~ om.m mo.o oa.o~ m.H o.om Homucoo
.m.m m .m.m m .m.m M .m.m M
mm>mH mmm mm>w~ mo Ea mmm am No AHE\aEV Ahmo\ax\aev
hum>muom ucmucoo mmoo ocm
axm mo mums: ammmmommm mammuomm Hmmmmume 9mm?

 

.Esummmumom mxmmz mmmzu mmmm admumuomm cw >uw>mmom Axmv mmmcwx
mum>sm>m ocm ucmucoo cmmuomm Omumamn co Hocmnmomodnomucmm mo mommwm ca mmnme

48

Liver Weight (Table 17), Protein Concentration, and
Activity of Pyruvate Kinase in the Pups (Table 18)

Pups from seven treatment groups all had similar liver
weight. Protein concentration was lower in the pups from
dams treated with technical grade PCP than those treated with
purified PCP. Pups of the dams receiving low doses of
technical grade PCP showed the greatest decrease in protein
concentration among all three groups treated with technical
PCP. The mean (28.1 mg/ml) was significantly different from
the control (34.1 mg/ml) at the level of 0.05. Units of
pyruvate kinase per gram of liver or per liver was similar
among all the groups. The specific activity of pyruvate
kinase tended to be higher in the pups from the technical
grade PCP treated groups than the purified PCP treated groups
(p < 0.1) .

Phase III:

Reproductive Performance of the Offspring

All but one of the female rats were bred within the first
estrual cycle. The exception was from the high dose level of
technical grade PCP treated dams, and was bred on day 10 after
being paired. A few bred rats were found not to be pregnant
on day 21 of gestation when the rats were sacrificed. The
number of non-pregnant rats relative to the number of obser-
vations in each treatment is presented in Table 19. No

statistical significance was observed.

49

Table 17 Liver weight of the female offspring3

from the dams receiving PCP treatments
during gestation.b

 

Test material

 

and dose Liver weight (gm)
(mg/kg/day)
i 5.2:.
Control 10.7 0.4
Pentachlorophenol,
purified
0.34 10.3 0.2:
3.40 10.2 0.4
34.00 10.7 0.3

technical grade

0.40 11.0 0.2
4.00 . 9.9 0.2
40.00 11.1 0.3

 

a Data was collected when the offspring were

eight weeks old.

b There is no statistical difference among the

treatments.

50

.om>mmmbo mmm3 mmocmmmwmmo acmommmcamm 02 a
.Nc.ouv Q .QUQ momma Hm0mcsomu mo

szoma 50mm mumo omHOOQ cmzu ucmmmmmmo mm QOQ omwmwmsQ mo szoma 50mm mumo omHOOQ mm

.m.o v o .moo momma moomoeoom mo

szoma Eomm mumo omHOOQ cmzu ucmmmmmmo mm QUQ omfimmmsQ mo szoma 20mm mumo omHOOQ om

 

 

 

.mmumcmaOEo: mm>wm mo HE me ammuomQ no as n
.oao mxmm: m mmm3 acwQOmuo may cmz3 omummaaoo mm3 mumo m
va med o.a m.va mc.o oam.e~ m.H Ma.mm oo.ov
Hm «NH H.H m.~H vo.o omm.m~ ma mv.m~ oo.v
m «mm m o a ma No a oNN on m mama mm ow o
momma Hmowczomu
ca QMH m.o h.~H mo.o mom.- m.m m~.vm oo.vm
om Hva m.m a.mm Ho.o mam.v~ m.~ mm.~m ov.m
AH mam H.H v.~a No.o 0mm.am m.c ma.vm em.o
ommmmmsQ
.Hocchomoflcomuch
Ha vma c.c v.v~ No.o mh.v~ m.o H.vm Homucoo
.m.m m .m.m m .m.m M .m.m M
AcwmuomQ up
mm>m~ me mm>ma mo Ea me Ea me muflczv AaE\aEV Azmo\ax\aev
2Q mo hum>wuom ucmmcoo mmoo ocm
A:d2\mHOE :V xQ «0 mafia: mammoQO ammuomQ Hmmmmume umme

a

 

fl

.COMumumma aCmmso mucmEummmu QOQ adm>flmomm mEmo mam Eomw acwQOmmo
mamemw mzu am >mm>muom AMQV mmmme mum>sm>Q ocm acmucoo :mmuomQ owqumz

ma manmfi

51

Table 19 Fertility of the offspring from the dams
treated with pentachlorophenol.

 

 

Test material No. of No. of
and dose non-pregnant bred rats
(mg/kg/day) rats
Control 0 12
Pentachlorophenol,
purified
0.34 1 9
3.40 l 7
34.00 1 3

technical grade

0.40 l 8

 

52

No significant changes were observed in terms of
maternal body weight gain, litter size, sex ratio of
fetuses (Table 20), and implantation rate (Table 21).
However, the number of resorptions was lower for the
offspring from the PCP treated groups than that from the
control group (Table 20). Rats from the dams receiving low
dose levels of technical grade PCP showed the lowest number
of resorption sites which was significantly different from
the control value, p <:0.01.

All of the fetuses that were recovered had survived

for longer than fifteen minutes.

53

.mmmsumm ammo» mo

.0: TSU On. 0>HUMHQH mmmflumm THME H0 .02 OER.

.COmumumma «0 am zmo on o amo some

om mmmumEmme mzu mew om>mmmno mmmB mmocmOmwmcamm Hmomumwmmum oz

.msam> menu mew cemum>mmmno mco

.omooo omen

 

 

HH.o mv.c H.o ~.c m.~ h.vH vH mam oo.ov
ma.o vm.o v.o 5.9 m.m «.ma mm mm oo.v
H~.o mm.o m.o ~.o o.H h.va mm mmm oe.o
momma Hmmmcnomm
no.c mm.o o.H o.m H.N m.NH o mama oo.vm
Hm.o om.o m.o m.o m.a a.ma om Nam ow.m
mo.o m¢.o m.c m.o m.a m.vH Hm mam wm.o
oommmooo
.HocanomoHnomuch
mo.o mm.o m.o m.H m.m m.ma mm wad Homucoo
.m.m m .m.m m .m.m m .m.m m
mmmmm .Eav :wma Ahmo\ax\aEv
memmmm xmm COAuQmOmmm mumm unammB mmoo ocm
mo .02 mmummq ommcmmumz Hmmmmume mmme

 

o.a:memuuo may no mocmEmOMme m>fluosooQOm mam
co cofimmumma acmmso HocszomoHnomuch Om mmsmOme mo mommwm

om mHnmE

54

Table 21 Implantation rate of the bred rats from
the dams treated with pentachlorophenol
during gestationa.

 

Test material

 

 

 

and dose Implantation rateb
(ms/kg/day)
SE 5.5.
Control 83 7
Pentachlorophenol,
purified
0.34 88 . 4
3.40 75 8
34.00 74 0
technical grade
0.40 79 7
4.00 79 2
40.00 84 6
a No significant difference were observed.
No. of Fetuses + No. of Resorption Sites X 100%

No. of Corpora lutea

DISCUSSION

Purified PCP had more effect on the reproductive
performance in the rats than the technical grade PCP,
namely, maternal body weight gain, number of fetuses carried
through gestation, number of resorption sites, weight of
fetuses, and sex ratio. Mortality of newborn pups and
activity of pyruvate kinase in the liver of lactating rats
were also affected more by the purified PCP than the tech-
nical grade PCP.

Because of the concern for the toxicity of dioxins and
furans, it has been suggested that more pure product should
be manufactured by the chemical companies than is now
supplied by most manufacturers. However, data from the
present study does not encourage this suggestion.

The experimental results from our study and from Dow
Chemical Company's study (50) are summarized in Table 22.
Larger numbers of replication for each treatment in Dow
Chemical Company's study than in ours probably contributes
to the higher degree of confidence in reporting the toxic
effects of both chemicals. Impurity content of the products
used in these two studies were somewhat different, although
in both studies the purified PCP had far less nonphenolics
than the technical grade PCP. Compared with the chemicals
used in the present study, the technical grade PCP used in
Dow's study had a higher content of octachlorodibenzo-p-

dioxin while the purified PCP had less content of all the

55

56

 

ommsmmme mo:

ommommmE mo:

mmmameocm
Hmcmmmxm Hmme

mmmsmmm mo
0mmmm xmm ommmmmocH

roooom
Qanmlc3omm ommmmmomo

mammoz moon
mmmmu ommmmmom:

mNmm
mmuumm ommmmmoma

mmmmm
cememOmmm ommmmmOCH

cemmmmmma acmmso
mxmmCm ommm Hmcmmmmz

:Hma unawmz >oon
HmcmmmmE ommmmmomo

 

momma
ommummsQ HmOwcnomB

 

.D.m.2

momma
ommmmmsQ HmOmcnomB

 

300

mucmsmmmmu moo mo mommmm

 

.nm>mmmmm>mc: mumum cmamnomz ocm
>chEOU HmOmEmzo 30a Eomw mumsmmm HmmcmEmmexm mam mo >mmEESm mm mabme

57

 

ommsmmmE

ommsmmme

ommsmmme

ommsmmmE

ommsmmme

ommsmmmE

ommsmmme

mo:

mo:

m0:

m0:

uoc

mo:

mo:

.ommmaamum

mo ommsm .COMmmOMmmmmo
mo mmmmcmo ommsmc: mo
omxmmmo .>mmmmescmesmv
mmmnmcmmmm

ACOmmmommmmmo
omamHmo .QOnm HmEmocnm
.ammmmescmesmv mmmnmumm>

mommsm mo mmnesa
.hmmmmfiscmesm. mnmm

mmst mmbesm

Acoflumommmmmo
oomomoo. mmoxm

"mmmHmEocm Hmmmmmxm
mmmmmm: ommmamo
mEmom msomcmmsonsm

"mmmsumu mo
mmmHmEocm mmsmmmm muom

 

momma

ommummsQ Hmomcnome

 

030m.

2

momma
omwmmmsQ Hm0mczoms

 

300

mucmEummmB QUQ NO mommwm

 

Ao.u:OOV NN mHDmB

58

 

+ I ommsmmme mo: omo mxmmz

03m 0m summn 80mm

humammmoe ommmmmOCm
"cemummmma acmmso QOQ numz ommmmmm
mEmo mnu Eomu cemmmmmcma mmmwm
mam mo mocmEmOMme HmmmcmmOQ
+ I ommsmmme mo: mmmm
acwmmuomm mzm mo >mm>mmom
mmmme mum>zm>Q mmmemz
+ I ommommmE mo: mmmm aCHDmmomm mam mo
mcmucom cmmmomQ mmmemz
I I ommsmmmE mo: mmmm acmmmuomm
mo unammB mm>mq
I I ommsmmmE mo: mumm
mcmcame ms» mo >mw>mu0m
mmmcmx mum>sm>Q mmmemz
I I ommsmmme mo: mumm mcmcame mnm mo
mcmmcoo cmmmomQ mamem:
I I ommsmmme mo: mumm mcmcame
mo unamm3 mm>mq

momma momma

ommmmmsQ HmOmcsomB

 

.D.m.2

ommmmmsQ Hm0mccome

 

BOO

mucmEummmm QUQ HO momwmm

 

1o.moooc mm 0moos

59

 

I I ommsmmms mo: cOmmmmmma acmmso
cmma unammz >oon

I I ommsmmmE mo: omo
mxmm3 ucamm mm >mmmmmmmm

I I ommsmmme mo: omo mxmmB unamm mm >mm>mmom
mmmme mmm>sm>Q 0mmem:

I + ommsmmme mo: oao mxmm3 usamm um mcmmcoo
cmmmomQ UHmem: ommmmmomo

I I ommsmmme mo: omo mxmm3 ucamm
mm usaflm3 mm>mm

I I ommsmmmE mo: oao mxmm3 unamm om acmcmmz
50mm unamm3 >oon

I I ommsmmme moc omo mxmm3 unamm om ammcmmz
Eomm mxmmcm ommm

I I ommsmmme mo: . ammcmmz mm
. utomos moon

+ I ommsmmme mo: acmcmmz mm
0mmmm xmm ommmmmUCm

 

momma momma
ommmmmoQ Hmomcnome ommmmmsQ HmOmcnome

 

 

m :mE mmm 0 mm a
.D.m.z 300 m m B QOQ m m mmm

 

1m.ucoov mm mmnoe

60

.moo mommmmoo moo norm
ommsmmme mmmmEmme ms» :0 mommum mmmmmma om: QOQ momma HmOmcsome

o
.QUQ momma Hm0mccomm
mam cmcm ommsmmme mmmmEmme ma» :0 mommmm mmummma om: QOQ ommmmmsQ o
.om>mmmno
mm: mommum mcmOHHmcamm >Ham0mmmmmmmm oc mmzm mmmmOmocm =I= mone>m b
.om>mmmno mm: macmno mcmOmwmcamm mmnu mmmmOmocm :+= mone>m m

 

I I ommsmmme mo: mmmm cemmmmcmHQEm

 

momma momma
ommmmmsQ mmomcnome ommmmmsQ mmOmcsome

 

 

m :mE mmm 0 mm
.D.m.2 300 m m B QUQ m m mmm

 

.m.m200. mm omnoe

61

nonphenolics determined. The route of dose administration
was different in the two studies; oral in Dow's study and
intraperitoneal in the present study. However, both studies
showed higher toxic effects of purified PCP on fetal develop-
ment than that of technical grade PCP.

Compared with technical grade PCP, purified PCP has
lower concentrations of nonphenolics. In the purified PCP,
there are no impurities present other than what has been
reported in the technical grade PCP. It seems that the high
concentration of the impurities present in the technical
grade PCP minimizes the toxicity of pentachlorophenol.

There was no evidence supporting either theory that the
toxic effects of PCP were caused by the parent compound or
by its metabolites; therefore, it is difficult to propose
the mechanism through which the nonphenolics cause their
detoxification effects.

Ahlborg and Thunberg (53) reported that dechlorination
of PCP in rats was enhanced by pretreatment with 2,3,7,8-
TCDD. The dechlorination was found to be mediated by liver
microsomal enzymes which metabolize PCP to form products
less toxic or more water soluble. Although 2,3,7,8-TCDD
was not detected in any of the PCP samples, it is generally
believed that all other isomers of PCDD have the same effects
on the drug metabolism system as 2,3,7,8-TCDD.

Although Larsen et a1. (51) has concluded that the
amount of placental transfer of PCP is negligible in rats,

the present study shows that PCP greatly affects the weight

62

and survival of fetuses and newborn pups from dams treated

with high doses of purified PCP during gestation. Four

possible interpretations are given for this phenomenon.

(l)

(2)

(3)

(4)

In Larsen's study doses were given orally while
in the present study doses were given intra-
peritoneally. Larger quantities of PCP might
have reached the placenta when it was adminis-
tered intraperitoneally rather than orally.

The compound might be extensively accumulated
in the fetuses after the mutiple-treatment was
given to the dams.

It may not be necessary for PCP to act directly
on the fetuses to cause toxic effects. A non-
detectable level of toxic chemical in the
fetuses gives no assurance of non-effected
offspring by the chemical.

A very small amount of PCP exposure may cause
extensive effects on the fetal and neonatal

performance.

In the treatment groups receiving high dose levels of

purified PCP, sex ratios of the fetuses recovered from

cesarean section and of newborn pups surviving to weaning

were higher than those in the control group. Fewer female

fetuses and pups survived than males. Body weight of the

female fetuses in the same treatment group decreased 20%,

while the male's weight was not affected as much. Both

parameters, sex ratios and fetal weight, indicated that

63

during the early developmental stages the female offspring
are more susceptable to the purified PCP than the male
offspring.

Body weight of the newborn pups was taken as the average
per litter; therefore, body weight of female pups was not
available until after weaning when pups of different sex were
separated. If the decreased weight of near-term fetuses is
an indication of the decreased birth weight of pups, female
pups from the high dose level of purified PCP treatment group
should be born with less body weight than the others.
However, by the 28th day when the first value of body weight
was available for the female pups, no differences were
observed among any treatment groups. The catch-up of body
weight by the female pups must have occured during their
first twenty-eight days of life.

The uptake and release of nutrients by the placenta is
an energy dependent process (54). There is abundant evidence
for the active transport of nutrient materials from maternal
to fetal circulation; the substances which have been proved
include iron, amino acids, lipids, cobalamin and calcium (55).
Metabolic uncouplers decrease the uptake and release of some
amino acids in the placenta (55). It would not be surprising
if future research were to reveal that uncouplers also
suppress active transport of other materials.

Pentachlorophenol is a powerful uncoupler of oxidative

phosphorylation; hence, the intrauterine growth retarded

64

offspring mentioned priviously may be caused by decreased
nutritional supply from the maternal tissue. The catch-up
growing pattern for growth retarded animals is a typical
model of moderate malnutrition during the developing period.
Impaired nutrient transport function may also be the cause
of high resorption rates and small litter size in the same
kind of treatment groups.

Pentachlorophenol as an uncoupler of oxidative
phosphorylation increases mitochondrial respiration and
fumarase activity in animals (27).. Under this condition
glycolysis is expected to increase to supply substrates
for the TCA cycle. In this study increased glycolysis
is reflected in the increased activity of hepatic pyruvate
kinase in the lactating rats receiving the treatment of
purified PCP. The increased activity of pyruvate kinase
was also observed in the pregnant rats receiving the medium
and the low doses of purified PCP and the high and the
medium doses of technical grade PCP, although the statistical
evidence was not very strong.

Pyruvate kinase is normally found in the liver in both
its active and inactive form. Unless the animal has a need
for a high rate of glycolysis, the demand for moderate
increase in activity of pyruvate kinase can be filled by
conversion of the inactive to the active form. Since the
assay measures total amount of pyruvate kinase including
both. the active and inactive forms, it cannot detect the

conversion between the inactive and active forms in vivo.

65

Based on the results from this study, it is proposed that
PCP increases the synthesis of hepatic pyruvate kinase in
lactating rats. However, the data are not as convincing

for the pregnant rats. According to Romsos et al. (57),

the energy requirements for lactating rats is three times
that for pregnant rats. Also, the hepatic fatty acid
synthesis in the lactating rats is 3.8 times that for the
pregnant rats. Consequently lactating rats have a higher
rate of hepatic glycolysis than pregnant rats. In pregnant
rats treated with PCP, conversion of pyruvate kinase from
the inactive to the active form may be adequate to stimulate
glycolysis to meet energy needs. However, for lactating
rats the energy demands are normally so much greater that

an increase in total pyruvate kinase is necessary to support
a high rate of glycolysis, particularly when oxidative
phosphorylation is partially uncoupled by PCP. This may
explain the difference in the amount of hepatic pyruvate
kinase observed between pregnant rats and lactating rats
after being exposed to PCP.

If there is increased hepatic pyruvate kinase activity
in lactating animals environmentally exposed to low levels
of PCP, then this may be of major concern to dairymen. In
ruminant animals a very low amount of glucose is absorbed
from the intestines. The liver is the major organ to supply
glucose for metabolism, pregnancy, and lactation. Therefore,

for rapid gluconeogenesis it is important to keep the

66

activity of hepatic pyruvate kinase low so that phosphoenol
pyruvate will be available for gluconeogenesis rather than
oxidation. 3

It is generally believed that glucose availability is
important in regulating milk synthesis. The possible
increase in activity of hepatic pyruvate kinase in dairy
cows exposed to PCP would decrease gluconeogenesis in the
liver and consequently decrease milk production.

However, not much is known about the mechanism that
keeps the activity of hepatic pyruvate kinase low in the
dairy cow. PCP exposure may not increase the activity of
pyruvate kinase if there is a different control mechanism
in ruminants than in rats. Therefore, the high demand of
substrate for increased rate of TCA cycle caused by uncoupled
oxidative phosphorylation in the cow would probably be met
by increased oxidation of propionate. This would decrease
propionate availability for gluconeogenesis resulting in

low blood glucose and low milk production.

(l)

(2)

(3)

(4)

CONCLUSIONS

Pentachlorophenol at high levels did affect
reproduction in rats.

More toxic effects on reproduction were observed
in rats treated with purified PCP than in rats
treated with technical grade PCP.

Prenatal PCP exposure had no effect on the
reproductive performance of first generation.
Further study of PCP effects on dairy cows should
emphasize glucose metabolism, enzyme activity in
carbohydrate metabolism, milk production, birth

weight of calves and survival rate of calves.

67

BIBLIOGRAPHY

10.

68

BIBLIOGRAPHY

Armstrong, H.W., 1969. Pentachlorophenol poisoning
pin a nursery for newborn infants. II. Epidemi-
ologic and toxicologic studies. J. Pediat.,
75:317.

Barthel, W.F., A. Curley, C.L. Thrasher, V.A. Sedlak,
and R. Armstrong, 1969. Determination of
pentachlorophenol in blood, urine, tissue and
clothing. J. Assoc. Off. Anal. Chem., 52(2):
294.

Champman, J.B., 1970. Poisoning with PCP. Brit. Med.
J., 2(5707):480.

Baader, E.W., and H.J. Baver, 1951. Industrial
intoxication due to pentachlorOphenol.
Industrial Med. and Surgery, 20(6):2286.

Bergner, H., P. Constantinidis, and J.H. Martin, 1965.
Industrial pentachlorOphenol poisoning in
Winnipipeg. Canad. Med. Ass. J., 92:448

Bevenue, A., J. Wilson, L.J. Casarett, and H.W. Klemmer
1967. A survey of pentachlorophenol content in
human urine. Bull. Environ. Contam. Toxicol.,
2(6):3l9.

Wyllie, J.A., J. Gabica, W.W. Benson, and J. Yoder,
1975. Exposure and contamination of the air
and employees of a pentachlorophenol plant,<
Idaho - 1972. Pest. MOnitoring J., 9(3):150.

Buu-Hoi, N.P., P.H. Chanh, G. Sesque, MC. Azum-Gelade,
and G. Saint-Ruf, 1972. Organs as targets of
intoxication. Naturwiss., 4:174.

State of California, Dept. of Public Health, 1970.
Pentachlorophenol poisoning in the home.
Calif. Health, 27(12):13.

Kimmig, J., and K.H. Schulz, 1957. Berufliche akue
(sog. chlorakue) durch chlorierte aromatische
zyklische ather. Dermatologica., 115:540.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

69

Sharma, R.P., R.J. Kociba, and P.J. Gehring, 1977.
Immunotoxicologic effects of 2,3,7,8-tetra-
chlorodibenzo-p-dioxin in laboratory animals.
Abstract in: Symposium on pentachlorophenol,
Pensacola (F1.) (Ranga Rao, K. and Richard,
N.L. ed.), 266.

Gupta, B.N., J.G. Vos, J.A. Moore, J.G. Zinkle, and
B.V. Bullock, 1973. Pathologic effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin in labora-
tory animals. Environ. Hlth. Perspect., 5:125.

Harris M.W., J.A. Moore, J.G. Vos, and B.N. Gupta,
1973. General biological effects on TCDD in
laboratory animals. Environ Hlth. Perspect.,
5:101.

Kociba, R.J., P.A. Keeler, C.N. Park, and P.J. Gehring,
1973. 2,3,7,8-tetrachlorodibanzo-p-dioxin
(TCDD): Results of a lB-week oral toxicity
study in rats. Toxicol. Appl. Pharmacol.,
35:553.

Vos, J.G., J.A. Moore, and J.G. Zinkl, 1973. Effect
of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the
immune system of laboratory animals. Environ.
Hlth. Perspect., 5:149.

Vos, J.G., J.A. Moore, and J.G. Zinkl, 1973, Toxi-
cology of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) in CS7Bl/6 mice. Toxicol. Appl.
Pharmacol., 29:229.

Groth, W., 1976. Histopathologische Befunde beim
Zinkmangelsyndrom und nach anschlieBender
Ainkrepletion. Zbl. fuer Veterinaermed., 23:31.

O'Dell, B.L., P.M. Newberne, and J.E. Savage, 1958.
Significance of dietary zinc for the growing
chicken. J. Nutr., 65:503.

Vos, J.G., J.G. Kreftenber, H.W.B. Engel, A.
Minderhoud, and L.M. Van Noorle Jansen, 1978.
Studies on 2,3,7,8-tetrachlorodibenzo-p-
dioxin-induced immune suppression and
decreased resistance to infection: Endotoxin
hypersensitivity, serum zinc concentrations

and effect of thymosin treatment. Toxicol.,
9:75.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

70

Bucher, T., and G. Pfleiderer, 1955. Pyruvate from
muscle. Methods in Enzymol. 1:435.

Lowry, O.H., N.J. Rosevrough, A.L. Farr, and
R.J. Randall, 1951. Protein measurement with
the folin phenol reagent. J. Biol. Chem.
193:265.

Kociba, R.J., D.G. Keyes, J.E. Beyer, B.M. Carreon,
C.E. Wade, D.A. Dittenber, R.P. Kalnins,
L.E. Frauson, C.N. Park, S..D. Barnard, R.A. Hummer,
and C.G. Humiston, 1978. Results of a two-year
chronic toxicity and oncogenicity study of
2,3,7,8-tetrachlorodibanzo-p-dioxin in rats.
Toxicol. Appl. Pharmacol., 46:279.

Innes, J.R.M., B.M. Ulland, M.G. Valerio,
L. Petrucelli, L. Fishbein, E.R. Hart,
A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart,
M. Klein, I. Mitchell, and J. Peters, 1969.
Bioassay of pesticides and industrial chemicals
for tumorigencity in mice: A preliminary note.
J. Nat. Cancer Inst., 42:1101.

Digiovanni, J., A. Viaje, D.L. Berry, T.J. Slaga, and
M.R. Juchau, 1977. Tumor initiating ability of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and
Arochlor 1254 in the two-stage system of mouse
skin carcinogenesis. Bull. Environ. Contam.
Toxicol., 18:552. ‘

Poland, A.P. Toxicology of chlorinated dibenzo-p-
dioxins. J. Environ. Health Sci., 8:0540.

Beatty, P.W., W.K. Vaughn, and R.A. Neal, 1977.
Effect of alteration of rat hepatic mixed-
function oxidase (MFO) activity on the toxicity
of 2,3,7,8-tetrachlorodibanzo-p-dioxin (TCDD).
Toxicol. Appl. Pharmacol., 45:513.

VinOpal, J.H., and J.E. Casida, 1973. Metabolic
stability of 2,3,7,8-tetrachlorodibenzo-p-
dioxin in mammalian liver microsomal systems
and in living mice. Arch Environ. Contam.
Toxicol., 1:122.

Hay, A., 1978. Vietnam's dioxin problem. Nature,
271:597.

Hay, A., 1978. Dioxin source is safe. Nature,
274:526.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

71

Nishizumi, M., 1978. Acute toxicity of polychlorinate
dibenzofurans in CF-l mice. Toxicol. Appl.
Pharmacol., 45:209.

Schwetz, B.A., J.M. Norris, C.L. Sparschu, V.K. Rowe,
P.J. Gehring, J.L. Emerson, and C.G. Gerbig,
1973. Toxicology of chlorinated dibenzo-p-
dioxins. Adv. Chem. Ser., 121:55.

Vos, J.G., J.A. Moore and J.G. Zinki, 1974. Toxicity
of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
in C57Bl/6 mice. Toxicol. Appl. Pharmacol.,
29:229.

Price, R.H., Jr., and D.M. Victor, 1977. The fate
of pentachlorophenol in an aquatic ecosystem.
Abstract in: Symposium on Pentachlorophenol
Pensacola (F1.) (Ranga Rao, K. and Richards,
N.L. ed.), 5.

Murphy, N.B.K., D.D. Kaufman, and G.E. Fries, 1979.
Degradation of pentachlorophenol in aerobic

and anaerobic soil. J. Environ. Health, Sci.,
314 (l):l.

Weber, K. and W. Ernst, 1978. Levels and pattern
of chlorophenols in water of the Weser Estuary
and the German Bight. Chemosphere, 11:873.

Reiner, B.A., 1975. Microbial catabolism of penta-
chlorophenol. Ph.D. Thesis, Purdue University.

Braun, W.H., J.D. Young, G.E.Blau, and P.J. Gehring,
1977. The pharmacokinetics and metabolism of
pentachlorophenol in rats. Toxicol. Appl.
Pharmacol., 41:395.

Braun, W.H., G.E. Blau, and M.B. Chenoweth, 1977.
The metabolism / pharmacokinetics of~pentachloro-
phenol in man, and a comparison with the rat
and monkey model. Abstract in: Symposium on
Pentachlorophenol Pensacola (F1.), 135.

Hoben, H.J., S.A. Ching, R.A. Young, and L.J. Casarett,
1976. A study of the inhalation of pentachloro-
phenol by rats: Part V. A protein binding study
of pentachlorophenol. Bull. of Environ. Contam.
and Tox., 16:225.

Casarett, L.J., A. Bevenue, W.L. Yauger, Jr. and
S.A. Whalen, 1969. Observations on pentachloro-

phenol in human blood and urine. Am. Industrial
Hyg. Assoc. J., 30:360.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

Hoben, H.J., S.A. Ching, and L.J. Casarett, 1976.
A study of the inhalation of pentachlorophenol
by rats. IV. Distribution and excretion of
inhaled pentachlorophenol. Bull. Environ.
Contaim. Toxic., 15(4):466.

Keplinger, M.L., G.E. Lanier, and W.B. Deichmann,
1959. Effects of environmental temperature
on the acute toxicity of a numer of compounds
in rats. Toxicol. Appl. Pharmacol., 1:156.

Parker, V.H., 1958. Effect of nitrophenols and
halogenophenols on the enzyme activity of
rat-liver mitochondria. Biochem. J., 69:306.

Cremer, J.E., 1961. A comparison of the action of
triethyldin with other drugs on creatine
phosphate level in rat brain and diaphragm
preparation. Biochem.. Pharmacol., 6:153.

Buffa, P., E. Carafolli, and U. Muscatello, 1963.
Mitochondrial biochemical lesion and pyrogenic

effect of pentachlorophenol. Biochem. Pharmacol.
12:769.

Bostroj, S.L., and R.G. Johansson, 1972. Effects
of pentachlorophenol on enzymes involved in
energy metabolism in the liver of the eel.
Comp. Biochem. Physiol., 41B:359.

Krueger, H., 8.0. Lu, G. Champman, and J.T. Cheng, 1966.
Effects of pentachlorophenol on the fish,
Cichlosoma bimaculatum. Abstracts from the 3rd.
Int. Pharmacological Cong., S. Paulo - Brazil,
24 - 30 July 1966.

Hinkle, D.K., 1973. Fetotoxic effects of pentachloro-
phenol in the gloden syrian hamster. Toxico.
Appl. Pharmacol., 25:455.

Schwetz B.A. and P.J. Gehring, 1973. The effect of
tetrachlorophenol and pentachlorOphenol on rat
embryonal and fetal development. Toxicol Appl.
Pharmacol., 25:455.

Schwetz, B.A., P.A. Keeler and P.J. Gehring, 1974.
The effect of purified and commercial grade
pentachlorophenol on rat embryonal and fetal
development. Toxicol. Appl. Pharmacol., 28:151.

Larsen, R.V., G.S. Born, W.V. Kessler, S.M. Shaw, and
D.C. Van Sickle, 1975. Placental transfer and
teratology of pentachlorophenol in rats.
Environmental letters, 10(3):121.

52.

53.

54.

55.

56.

57.

58.

59.

60.

73

Edwards, M.J., 1968. Congenital malformations in
the rat following induced hyperthermia during
gestation. Teratologyr 1:173.

Ahlborg, U.G., and T. Thunger, 1977. Effect of
2,3,7,8-tetrachlorodibenzo-p-dioxin on the
in vivo and in vitro dechlorination of
pentachlorophenol. In: Environmental Science
Research, Vol. 12. Pentachlorophenol, edited by
K. Ranga Rao. Plenum Publishing Corporation, 127.

Kelman, B.J., 1978. Effect of toxic agents on
movements of materials across the placenta.
Federation Proceedings,-38(8):2246.

Miller, R.R., T.R. Koszalka, and R. Brent, 1976.
The transport of molecules across placental
membranes. In: The cell surface in animal
embryogenesis and development, edited by
G. Poste and G.L. Nichloson. New York:
Elsevier/ North Holland, 1976, p. 145-223.

Miller, R.R., T.R. Koszalka, B.M. Davis, C.L. Andrew,
and R.L. Brent, 1974. Placental transport of
creatinine and amino acid in the rat.
Teratologyr 9:428.

Romsos, D.R., R.L. Muiruri, P. Lin, and G.A. Leveille,
1978. Proc. of the society for Exp. Bio. and
Med., 159:308. ‘

Kitchin, K.T., and J.S. Woods, 1977. 2,3,7,8-
tetrachlorodibenzo-p-dioxin induction of aryl
hydrocarbon hydroxylase (AJJ) in hepatic
microsomes from female rats. Abstrat in:
Symposium on Pentachlorophenol Pensacola (F1.)
(Ranga Rao, K. and Richards, H.L. ed.), 179.

Chu, J.P., and E.J. Kirsch, 1972. Metabolism of
pentachlorophenol by an axenic bacterial
culture. Appl. Microbiol., 23:1033.

Chu, J.P., and E.J. Kirsch, 1973. Utilization of
halophenols by a pentachlorophenol metabolizing
bacteriam. Dev. Ind. Microbiol., 14:264.