Analysis for Cadmium Mortality
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Data Items

1 Gender        0 Female
                1 Male

2 Dose          mg Cd/kg body weight

3 Denominator   number injected

4 dead_30       number dead at 30 days (a)

5 dead_60       number dead at 60 days (b)

-------------
(a) rats administered CdCl2 nine times
    over a 30-day period

(b) rats did not receive additional CdCl2 
    to day 60.

Data extracted from 

Table 1 "Incidence of mortality for rats administered
multiple (chronic) injections of Cadmium" 

in article "Analysis for Cadmium Mortality"

by 

Excerpts from article follow:


Analysis for Cadmium Mortality

INTRODUCTION

Cadmium is a rare, ubiquitous trace element of uncertain 
biologic effects, discovered in 1878 by Strohmeyer and Herman 
(CEC, 1978), and found in trace amounts in nearly all 
foodstuffs and drinking water (Perry et al., 1976). The trace 
metal is considered an important environmental pollutant 
because it accumulates in many different organisms (Webb, 
1975).

Levels of cadmium have been increasing in the environment 
throughout the past two decades in highly industrialized 
areas, and the trace metal has been assigned third-order 
status in eight orders of priority in the United States 
Earthwatch Program (Jensen et al., 1975). The major sources 
of cadmium contamination in air include smelting and refining 
of zinc and lead ores (Beton et al., 1966), combustion of 
coal and oil (Ellis et al., 1979), and disposal of sewage 
sludge and waste products (Fassett, 1975). Exposure of 
workers to the fumes of cadmium oxide dust has been 
recognized as a cause of an acute and sometimes fatal 
pulmonary edema (Adamsson et al., 1979). The absorbed element 
has been shown (Fassett, 1975) to be excreted very slowly, 
with continuous elimination in the urine for long periods of 
time after exposure has ceased (Shank et al., 1977).

Few references to differences in cadmium mortalities in males 
and females appear in the literature. Yoshikawa (1976) 
compared mortalities of male and female mice of the same age 
(in weeks) to which equal amounts of cadmium were 
administered. He observed that female mice showed higher 
death rates than male mice, indicating a greater 
susceptibility of females to the acute toxicity of cadmium. 
Testicular necrosis (Gunn and Gould, 1970; Aoki and Hoffer, 
1978; Kotsonis and Klaassen, 1978) has been shown in male 
rats. Here, we employ the SAS probit analysis (SAS, 1979) 
against the log(dose) as described by Finney (1971) to 
compare cadmium dose-response of young male and female 
Sprague-Dawley rats.
 
METHODS

Female rats, Rattus norvegicus (Sprague-Dawley strain), were 
bred to young males of proven fertility to obtain the 
necessary number of experimental animals. This species and 
strain was selected from the Texas Woman's University animal 
colony because of its previous use as a standardized mammal 
in both radiation and heavy metal studies. At 23 days of age 
the litters were weaned and sexed. Randomization was 
accomplished by assigning a number to each rat and using a 
Table of Random Digits- (Finney, 1971) for designation to the 
specific group. Animals were kept under strict sanitary 
conditions, with the temperature of the room held at 24 +/- 
2¡C. A diurnal environment was provided by artificial 
lighting, consisting of a 12-hr light (6AM-6PM) 12-hr dark 
(6PM-6AM) cycle. Rats were maintained on Purina Rodent 
Laboratory Chow (Ralston Purina Co., St. Louis) with water ad 
libitum. All rats were 80 days old at the start of the 
experiment.

Cadmium chloride (Fisher Scientific Co., Dallas) was 
dissolved in sterile distilled-deionized water and 
administered intraperitoneally. Doses were based on the total 
body weight of each animal at the time of injection and the 
volumes werc adjusted to provide the desired doses. A 
solution of 4.0 mg Cd/ml of sterile distilledÑdeionized water 
was used as the standard. Controls receiv~d a like amount of 
stcrile distilledÑdeionized water.

Acute cadmium lethality: 
A total of 96 females and 112 males were each injected once 
with one of the following concentrations: 0, 2, 4, 5, 6, 8, 
16, or 24 mg Cd/kg body wt. Deaths were recorded during a 30-
day period.

Chronic cadmium lethality:
A total of 90 females and 90 males were each injected every 3 
days for 30 days for a total of nine injections with one of 
the following: 0, 1, 2, 3, 4, 5, 6, 7, 8, or 16 mg Cd/kg body 
wt for total cumulative doses of 0, 9, l8, 27, 36, 45, 54, 
63, 72, or 144 mg Cd/kg body wt. Deaths were recorded during 
the injection period and for an additional 30 days.

Statistical and computer analyses
LD50 values were determined using the computerized 
Statistical Analysis System (SAS) of probit analysis (SAS, 
1979) This system is particularly useful in analyzing dose-
response data since it is more accurate than the graphical 
methods currently in use and, in addition, provides fiducial 
limits with its estimates. In addition, the researcher needs 
only to supply the dose, the number of animals tested, and 
the number responding (dying) at each dose. Other advantages 
are that only a few minutes at the terminal are required for 
complete analysis and the required computer time is less than 
one second. The reader is referred to the SAS User's Guide 
(SAS, 1979) for computational details. a chi-square value was 
used to test the statistical validity of the procedure and 
Student's t test was used to compute the fiducial limits. 
Statistical comparison of LD values were made by using the 
bivariate regression as implemented by the Texas Women's 
University DECsystem 20 computer.

DISCUSSION

In the quantitation of heavy metal effectiveness it is useful 
to compute the median lethal dose (LD50) to the exposed 
population. The computerized Statistical Analysis System 
(SAS) of probit analysis is particularly useful in analyzing 
doseÑresponse data because it is more accurate than the 
graphical methods currently in use and, in addition, provides 
fiducial limits with its estimates. Generally, the LD values 
of the chronic study indicate an increase in ef~ectiveness of 
dose as time of injections was protracted. However, the exact 
reason for the increased susceptibility of males to cadmium 
is not clearly understood. Differences based on the greater 
animal size were eliminated by adjusting the dose for each 
individual body weight. A plausible explanation would be the 
well documented hemorrhagic necrosis (Gunn and Gould, 1970; 
Kotsonis and Klaassen. 1978; Kundomal, Morgan, and Hupp 
1982).

Webb and Verschoyle (197B) suggested that the testis, in 
common with the liver and kidney, has the capacity for 
inducing synthesis of cadmium-thionein. These same 
investigators also reported that a small cadmium dose (100 
micro g Cd/kg) raised cadmiumÑthionein levels in female rats 
and protected them against the effects of a normally lethal 
dose (4.5 mg Cd/kg). However, the protective effect began to 
wear off after 3 days, whereas both the increased content and 
capacity for the synthesis of cadmiumÑthionein were 
maintained. This suggests that preinduced metallo thioneins 
do not play a significant role in the protection against 
acute cadmium toxicity. In the present study, synthesis of 
cadmium-thionein may explain why experimental animals were 
able to tolerate more cadmium given as multiple doses at 
frequent intervals than when administered as a single 
injection. However, it does not explain the greater 
susceptibility of males to the heavy metal. Chen and Ganther 
(1975a, b) identified a unique protein of high molecular 
weight (30,000) in the testis which has not been detected in 
other tissues. They further reported that the protein has 
less affinity for cadmium-metallothionein but was associated 
with cadmium injury and sensitivity of the testis.

Wolkowski-Tyl and Preston (1979) observed that a single 
subcutaneous injection of cadmium chloride causes typical 
testicular hemorrhagic necrosis in NAW male mice, and that 
these animals make only metallothioneins and not the dimer of 
cadmiumÑthionein (CdÑbp-D). However, when NAW male mice were 
given the female hormone progesterone at a dose of 0.1 g/kg 
body wt prior to cadmium exposure, there was no evidence of 
hemorrhagic necrosis of the testis and, in addition, there 
was production of the dimer (Cd-bp-D). This finding by 
Wolkowski-Tyl and Preston (1979) strongly suggests the 
protective nature of the dimeric cadmium carrier synthesized 
after cadmium exposure in animals primed with the proper 
hormonal millieu (high levels of circulating progesterone). 
The data of our study, in conjunction with the findings of 
Wolkowski-Tyl and Preston (1979), may explain the greater sex 
specificity of cadmlum toxicity. The significance of the sex 
and hormonal milieu for the in vivo dose -response of higher 
organisms to heavy metals is an area deserving further 
investigation.